Abstract: This paper established a model of alpha amylase inhibitor screening in vitro, and researched the inhibitory effects and the dynamic characteristics of chlorogenic acid compounds extracted from Stevia rebaudiana hairy roots through this model.The optimized parameters of this model were as follows: the enzyme concentration, substrate concentration, reaction temperature, and reaction time were 1.25 U/m L, 0.05~ 6 mg/m L, 37 ℃, and 30 minutes respectively. The inhibitory effect of Stevia rebaudiana hairy roots ( ACCC10060, R1601, A4) extracts on α-amylase was studied by this modle, and it found that they all had strong inhibitory effect on α-amylase and the IC50 were 12.43, 18.310 and 21.084 mg/m L respectively. The results of HPLC showed that total chlorogenic acid extract of Stevia hairy roots ACCC10060 mainly contains chlorogenic acid, 3, 5-dicaffeylquinic acid and 4, 5-dicaffeylquinic, whose content was 6.88, 19.90 and 1.50 mg/g respectively.Enzyme inhibition kinetics test showed that all the three chlorogenic acid had strong inhibitory effects on α-amylase.The inhibitory effects on α-amylase between the Stevia rebaudiana hairy roots extracts and the mixture which contains three chlorogenic acid standard products according to the proportion of total chlorogenic acid extracts had no significant difference.The results above indicated that the chlorogenic acids of stevia hairy roots demonstrated the excellent inhibitory effect of α-amylase, and the main active components were chlorogenic acid, 3, 5-dicaffeylquinic acid and 4, 5-dicaffeoylquinic acid which were expected to developing into a natural, efficient postprandial blood glucose inhibitors.