At present, there is wide application of microbial lactases, and these enzymes are characterized with rich source of origin, high yield, low production cost, and short production cycle. This study was based on Streptococcus thermophilus GST-6 isolated from traditional dairy products with lactase producing capability. Firstly, key factors that affect lactase production were determined by single factor tests. Then, the fermentation conditions for lactase production by GST-6 were optimized by using the method of response surface analysis.Based on the optimum fermentation conditions, the crude enzyme was prepared. After salting out by ammonium sulfate fractionation, the purified lactase was obtained by DEAE-SepHarose fast flow ion-exchange chromatography and SepHadex G-75 gel filtration chromatography. Furthermore, the kinetic properties and stability of the enzyme were studied. The results showed that key factors that affected lactase production were pH, inoculation rate and lactose concentration of the culture medium, and these factors had significant interactions. The optimum fermentation conditions of the culture medium were as follows:pH 6.78, inoculation at 1% and lactose concentration of 25.00g/L. The enzyme activity reached 112.63U/mL.The characteristics of the enzyme were determined as follows:optimum saturation ammonium sulfate fractionation by 10%～50%; maximum hydrolytic activity at pH 6 and temperature 30~40℃, Mn²⁺ could active enzymatic activty while Cu²⁺ could inhibite enzymatic activity. The enzymatic inhibition by SDS, galactose, and EDTA was a decreasing order. The K_m value and V_max of this enzyme was 0.27mmol/L and 2.20μmol/min.