Objective To prepare reference materials for detection of Cryptosporidium parvum nucleic acid according to procedures and processes for preparation of reference materials specified in the national criteria GB/T 15000 Directives for the work of reference materials. Methods A large number of oocysts of the C. parvum C1HN strain (subtype IIdA19G1) were obtained through calf passage, and purified, and genomic DNA was extracted from C. parvum oocysts using DNA extraction kits. Then, the source of oocyst DNA was traced. Once the traceability of the extracted DNA was verified, DNA samples were aliquoted and cryopreserved. The homogeneity, freeze-thaw stability, and short- and long-term stability of C. parvum oocyst DNA samples were evaluated with nested PCR and quantitative Real-time PCR (qPCR) assays, and the constant values were determined collaboratively by multiple institutions. All statistical analyses were performed using the software SPSS 27.0, and differences of means were tested for statistical significance with one-way analysis of variance (ANOVA) and paired-samples t test. Results The PCR amplification fragment was approximately 830 bp in size, which was consistent with the results detected by the national standard. Sequencing revealed that the SSU rRNA gene fragments on chromosomes 2, 7, and 8 of this strain exhibited 99.65% sequence identity with those of the C1HN strain, indicating complete concordance with the infecting strain, and the subtype was identified as ⅡdA19G1. Homogeneity tests showed that the Ct value of the samples was 15.00 ± 0.36, with a mean square of 0.17 between groups and 0.11 within groups, indicating no statistically significant difference (F = 1.49, P > 0.05). Freeze-thaw stability tests showed that the Ct values of the samples were 15.67 ± 0.25, 15.68 ± 0.22, 15.69 ± 0.27, 15.71 ± 0.27 on days 0 (t = 0.00, P > 0.05), 3 (t = 0.31, P > 0.05), 7 (t = 0.50, P > 0.05), and 14 following repeated freeze-thaw cycles (t = 0.71, P > 0.05), respectively. Short-term stability tests showed that the Ct values were 16.48 ± 0.28, 16.48 ± 0.25, 16.49 ± 0.28, 16.50 ± 0.25, 16.53 ± 0.26, 16.56 ± 0.25, 16.57 ± 0.24 following storage at 4 ℃ for 0 (t = 0, P > 0.05), 1 (t = 0.04, P > 0.05), 2 (t = 0.34, P > 0.05), 3 (t = 0.46, P > 0.05), 4 (t = 1.56, P > 0.05), 5 (t = 1.60, P > 0.05), and 10 days (t = 1.84, P > 0.05); 16.20 ± 0.18, 16.21 ± 0.14, 16.21 ± 0.13, 16.22 ± 0.14, 16.23 ± 0.14, 16.24 ± 0.14, and 16.25 ± 0.15 following storage at 25 ℃ for 0 (t = 0.00, P > 0.05), 1 (t = 0.17, P > 0.05), 2 (t = 0.28, P > 0.05), 3 (t = 0.47, P > 0.05), 4 (t = 0.61, P > 0.05), 5 (t = 1.09, P > 0.05), and 10 days (t = 1.40, P > 0.05); and 16.21 ± 0.17, 16.22 ± 0.16, 16.23 ± 0.16, 16.24 ± 0.17, 16.26 ± 0.18, 16.25 ± 0.18 and 16.26 ± 0.19 following storage at 37 ℃ for 0 (t = 0.00, P > 0.05), 1 (t = 0.33, P > 0.05), 2 (t = 0.56, P > 0.05), 3 (t = 1.10, P > 0.05), 4 (t = 1.20, P > 0.05), 5 (t = 1.34, P > 0.05), and 10 days (t = 1.89, P > 0.05), respectively. Long-term stability tests showed that the Ct values were 15.67 ± 0.11, 15.67 ± 0.21, 15.67 ± 0.21, 15.67 ± 0.25, 16.67 ± 0.24 and 15.67 ± 0.23 following storage at -20 ℃ for 0 (t = 0.00, P > 0.05), 30 (t = 0.01, P > 0.05), 90 (t = 0.11, P > 0.05), 180 (t = 0.23, P > 0.05), 270 (t = 0.33, P > 0.05), and 360 days (t = 0.39, P > 0.05), respectively. Nested PCR assays of samples stored for 180 and 360 days amplified specific bands with approximately 830 bp in size, and sequencing results were consistent with those previously obtained. All participating institutions provided verification reports confirming that the prepared reference materials might be used as a qualitative positive control for detection of C. parvum nucleic acid. Conclusion The prepared reference material meets the requirements specified in the national standard GB/T 15000 Directives for the work of reference materials with reliable traceability, good homogeneity, stability in compliance with specifications, and accurate constant values.