Laboratory studies were conducted to examine the effect of pH, moisture, temperature, and substrate concentration on the activity of soil L-glutaminase in three black soils and three red soils, Telangana, India. The optimum pH for NH4+-N released by L-glutaminase activity in soils was 8. The L-glutaminase activity rose with increase in moisture level from 10 to 50% when the soils and later showed a decreasing activity. An increase in temperature increased the L-glutaminase activity from 10 to 50 °C and decreased sharply with a further increase in temperature. The temperature beyond 50 °C, Q10 values were <1, indicating the deactivation of L-glutaminase. Ea values varied from 28.72 to 40.17 kJ mol−1. The L-glutaminase activity increased with the increase in substrate concentration from 2 to 40 mM and a negligible increase in catalyst activity was noticed at higher concentration. Application of three linear transformations of the Michaelis–Menten equation showed that the apparent Km values of L-glutaminase in six soils ranged from 13.85 to 46.00 mM and the apparent Vmax values ranged from 9.55 to 47.62 μg NH4+ mineralized g−1 soil 4 h−1. Montmorillonite clay and higher O.C.% make black soils have higher Km and Vmax than red soils. Black soils may have longer-lasting enzymatic activity but lower efficiency due to lower substrate accessibility; such slow organic matter decomposition leads to longer soil health maintenance.