[Objectives] Vigilance behavior is a kind of animal response to external risks that can detect or avoid dangers in advance to improve survival rate, when it occurs, the distance between animals and external risk source is defined as vigilance distance. Habitat changes and human disturbance caused by rapid urbanization have a significant influence on the vigilance behavior of wildlife. The vigilance distances of populations adapted to urban environments involves changes in behavior and physiology, reflecting phenotypic plasticity or evolution. The objective of this study was to explore the relationships between vigilance distance of Microhyla fissipes and the changes of habitat and human activity intensity caused by urbanization along an urbanization gradient (urban-suburban-rural). [Methods] In this study, we selected M. fissipes as study species, and measured 100 M. fissipes male individuals (urban: 15 males; suburban: 51 males; rural: 34 males) from 22 study sites (urban: 3 sites; suburban: 10 sites; rural: 9 sites) along an urbanization gradient (urban-suburban- rural) between May and August in 2020 and 2021 in Shanghai, China. We used the percentage of impervious surface (including buildings and roads) in each 2 km-radius landscape to represent the urbanization index of these study sites. In addition, we also collected the human population density of each study site to represent human activity intensity. Then we tested the normality of vigilance distance using the Shapiro-Wilk test, and general linear models were used to test the effects of urbanization index and human population density on vigilance distance of M. fissipes in each study site. [Results] We found that the vigilance distance of M. fissipes ranged from 2.8 m to 5.1 m (mean 3.9 ± 0.5 standard deviation) (Table 1). By ranking all possible candidate models based on their Akaike’s information criterion corrected (AICc), urbanization index was the most important predictor in the best model (ΔAICc < 2) (Table 2). Model average coefficients showed that vigilance distance of M. fissipes was significantly negatively correlated to urbanization index (P < 0.01) and also negatively related to human population density, although not significant (P > 0.05) (Table 3). [Conclusion] Our founding suggested that amphibians change the vigilance distance in response to urbanization, which benefits us to understand the response mechanism of amphibian behavior to urbanization.