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A large part of India is covered with Black Cotton soil which is clayey in nature. These are the soils of residual deposits formed from basalt. In this research, bio-enzymes a chemical plasticizer and their effect on the engineering properties of soil are discussed. Recently there are many bio-enzymes are available for soil stabilization. Here we used biochemical enzyme-ECOZYME AE-40 in soil stabilization. The black cotton soil sample was collected from Kovilpatti, Tamil Nadu. The soil samples are treated & tested in the laboratory. The results of consistency limit, unconfined compression test, free swell index, Proctor compaction test CBR test indicates the properties of soil, which are important for the pavement construction. In this CBR test value is considered while designing flexible pavement. The treated and untreated soil condition is should vary among laboratory investigation in flexible pavement design (IRC: 37-1970