The impact of transgenic Bacillus thruingiensis (Bt) cotton cultivation on soil enzymes and physico-chemical parameters of soil were investigated. Soil samples were collected from surface (0-20 cm depth) of agricultural fields near Bathinda, District. Where Bt cotton (RHC134) and non Bt cotton variety had been continuously cultivated for last two years. Soil samples were collected after harvesting of cotton crop. A control sample was collected from the adjoining waste land where no crop was grown from last many years. To observe effect of Bt cotton on soil biochemical properties, activities of soil enzymes such as amylase, cellulase, urease, dehydrogenase, alkaline phosphatase and acid phosphatase were assayed. Statistically significant enhancement in activities of the above enzyme was observed in Bt cotton soil samples as compared to non Bt cotton soil samples. No difference was observed in cellulase activity between Bt and non Bt cotton soil samples. Further, to study the effect of Bt cotton on physico-chemical properties, pH, conductivity, texture, total organic carbon (%) and organic matter (%), available nitrogen and available phosphorous content were estimated. In conclusion, significant changes were observed in Bt cotton grown soil samples.