Background: Considering the importance of Cryptosporidium spp. to public health and the country's economic situation due to the threat of an outbreak and economic losses. Objectives: this study aimed to detect Cryptosporidium spp. in human in Babylon province using traditional and molecular techniques. Methods: this study was conducted in Babylon province from September, 2023 to March, 2024.a total of 100 human stool samples were examined microscopically for detection of Cryptosporidium spp. from both diarrheal and non-diarrheal patients attend hospitals and public health centers laboratories. Results: by using microscopic examination. The rate of infection with Cryptosporidium spp. was (25/100), 25%. According to sex, the highest infection rate in humans was in males (17/59),28.81% compared to females (8/41) ,19,51%. was recorded in the rate of infection between age groups, the highest rate (13/28), 46.42% observed in < 5years of human. Rural area revealed the highest rate of infection than urban area in human (16/44),36.63%. In molecular diagnosis, DNA extraction was performed for 50 stool samples human 28% (14/50) C hominis 10% (5/50) C parvum 18% (9/50) by nested PCR, in order to investigate the genotypes of Cryptosporidium spp. from human in Babylon, five Cryptosporidium spp. isolates. were genetically characterized by SSU-rDNA gene sequencing. The results showed C parvum (1/5) C hominis (4/5). Conclusion: these findings suggested that infection of humans by zoonotic genotypes were the most common genotypes detected in all human samples in Babylon province, to enhance the effectiveness of the One Health approach in preventing Cryptosporidium spp. infection, it is crucial to have a deeper understanding of the environmental, epidemiological, and etiological aspects linked to Cryptosporidium infection. This knowledge will aid in the development of more effective risk management strategies. The future One Health plan seeks to incorporate diverse and interdisciplinary expertise.