To understand the prevalence of human hookworm infection in Yunnan Province, a total of 20 survey sites in 10 counties (cities) in Yunnan Province were selected by the stratified cluster random sampling method in 2015, according to the National Survey Plan for the Status of Key Human Parasitic Diseases and the implementation guideline. In each site, no less than 250 permanent residents aged 1 year and above were surveyed. Fecal samples were collected from the participants for examining, hookworm eggs using modified Kato-Katz method. The hookworm species was identified by the in vitro filter culture method. A total of 5 067 residents were examined, with an infection rate of 9.0% (457/5 067), among them, 94.5% (432/457) were severe, 2.8% (13/457) moderate and 2.6% (12/457) mild infection. Species identification was performed for 160 cases, of them, 139 was infected with Ancylostoma americanum, 16 with A. duodenale, while 5 with mixed infection. The infection rate was 8.4% (204/2 422) in males and 9.6% (253/2 645) in females. Highest infection rate was detected in the age group of 60 years and above (12.93%), in Dulong ethnic group (39.55%), in farmers (9.4%), and in the group of education level of primary school level (9.5%). The human hookworm infection rate was highest in the ecological area of southern part of Yunnan, Guangxi and Guangdong (17.14%), with Hekou County in particular having infection rate up to 30.6%. Human hookworm infection rate was 0.8% (6/759), 13.6% (311/2 287) and 6.9% (140/2 021) in regions with high, medium and low economic levels, respectively, with significant differences between the regions(P < 0.01). In conclusion, the human hookworm infection rate remains high in part of Yunnan Province, efforts should be made to continuously strengthen prevention and control measures in farmers, elders, and strait through ethnic minorities.