Objective To establish a rolling circle amplification (RCA) method for detection of Cysticercus pisiformis infection in rabbits. Methods The novel-miR1 derived from C. pisiformis in rabbit serum served as the diagnostic target to establish an RCA diagnostic method for cysticercosis pisiformis fection in rabbit. To establish the RCA method, ligation sequence and the locking probe sequence were designed, and five important reaction conditions were optimized, including the ratio of ligation sequence and padlock probe, ligation reaction time, ligase dosage, amplification reaction time, and dNTP dosage. The sensitivity of the RCA method was assessed, the novel-miR1 standard was serially diluted into 9 samples of different concentrations ranging from 1 fmol/L to 100 nmol/L. The sensitivity and specificity of the optimized RCA methods were assayed using the serum miRNA from 20 healthy rabbits and 20 C. pisiformis infected rabbits (Laboratory preserved samples), and analyzed by the receiver operating characteristic curve (ROC curve) method. To evaluate the application effectiveness of the RCA, 20 female rabbits were infected by gavage with 1 000 eggs of T. pisiformis for collection of serum every month post-infection, which were used to prepare miRNA for application in RCA method under optimized condition. Results The agarose gel electrophoresis results showed that the RCA amplification products remained in the sampling wells of agarose gel, forming a bright band. The tests to optimize RCA condition indicated that the concentration of ligation sequence was 2 μmol/L, padlock probe was 1 μmol/L (ratio of ligation sequence and padlock probe was 2∶1), T4 DNA ligase was 350 U, and the efficacy of ligation reaction was found the highest when ligating for 180 min. The optimal amplification reaction system was 0.5 μmol/L dNTP and amplification reacted 240 min in 100 μl amplification reaction system. Lastly, the RCA method limit of detection was proved to be 10 pmol/L. The RCA method detected showed that the average serum miRNA fluorescence intensity of the samples from healthy rabbits and C. pisiformis infected rabbits were 53.298 ± 1.707 and 97.498 ± 5.892, respectively, which was statistically significant (t = 7.206, P < 0.01). ROC curve showed that the RCA method cut-off value was 61.69 and both sensitivity and specificity were 95%, the area under the curve (AUC) is 0.955 0, likelihood ratio was 19.00. Using the RCA method to detect 20 healthy rabbits’ serum miRNA found 19 samples were negative and 1 positive. The results of RCA detection of the C. pisiformis infected rabbits serum miRNA at different times found that 17 were positive, 2 were suspected, 1 were negative 1 month post-infection. One was negative, and 19 were positive 2 mouths post-infection. Three were negative, and 17 were positive 3 months post-infection. Conclusion An RCA method was established for detecting C. pisiformis infection in rabbits. It was demonstrated that novel-miR1 could be detected by RCA in the serum of rabbits within 3 months post-infection of C. pisiformis, showing good application potential.