Objective: To understand the etiological characteristics of hand, foot and mouth disease (HFMD) in Henan Province from 2021 to 2024, and provide baseline data for the development of more effective prevention and control strategies. Method: Data were collected from HFMD surveillance in Henan during 2021-2024. The positive rate of clinical samples and etiological characteristics of the pathogens were described, and multivariate logistic regression analysis was used to identify the influencing factors of positive rate. Results: From 2021 to 2024, a total of 18 275 clinical samples from HFMD cases were tested in Henan, the positive rate of enterovirus was 71.45% (13 057/18 275). Multivariate logistic regression analysis showed that higher positive rate was associated with being man, age 3 and 5 years, living in northern Henan, high incidence month, stool sample, and samples collected within 3 days after onset (all P<0.05). Among 9 338 samples tested for EV-A71, CV-A16, CV-A6, and CV-A10, the samples positive for CV-A16 accounted for the highest proportion (63.90%, 5 967/9 338), followed by the samples positive for CV-A6 (17.70%, 1 653/9 338), other enterovirus (15.16%, 1 416/9 338), CV-A10 (2.74%, 256/9 338), multi pathogens (0.28%, 26/9 338), and EV-A71 (0.22%, 20/9 338). The distribution of pathogens differed across years, age groups, case types, and areas (all P<0.001). CV-A16 was the predominant pathogen in 2021, 2022, and 2024, while CV-A6 was predominated in 2023. Younger age groups were mainly infected with CV-A16 and CV-A6, whereas older age groups were mainly infected with CV-A16 and other enterovirus. In mild cases 63.97% were infected with CV-A16, while in severe cases, 36.36% were infected with other enteroviruses and CV-A16 respectively. Although CV-A16 was the predominant pathogen in Henan, western Henan had the lowest proportion of CV-A16 infection cases (51.90%), while southern Henan had the highest proportion of CV-A16 infection cases (78.98%). Conclusions: The positive rate of clinical samples from HFMD cases in Henan was influenced by sex, age, area, month, sample type, and the interval between onset and sample collection. It is necessary to take targeted measures to improve the surveillance. Furthermore, the pathogen spectrum of HFMD in Henan continues to change, it is suggested to further improve the serotyping of enterovirus and conduct long-term and extensive surveillance for HFMD pathogen spectrum to understand the epidemiological patterns and incidence trends of HFMD.