Microsclerotia (MS) formation was successfully induced in Metarhizium rileyi (Ascomycetes: Hypocreales) in liquid culture. To optimise the process variables of liquid fermentation, we first used a two-level fraction design to confirm the variables, including inoculum density, initial pH, shaker speed, and temperature, affecting M. rileyi MS production. Three variables were found to be important. Subsequently, a 2³ full factorial central composite design (CCD) and response surface methodology were applied to ascertain the optimal level of each variable. A second-order polynomial was determined and shaker speed and inoculum density were found to be the primary variables affecting MS yields. Finally, we realised and optimised M. rileyi MS submerged fermentation based on previous findings. A maximum MS yields (3.84 × 10⁴ MS/mL) were recorded in submerged fermentation at an initial pH of 5.5, growth temperature of 26℃, inoculum density of 10%, higher aeration rate (150 rpm in the initial 3 days and 200 rpm in the subsequent 3 days), and higher agitation rate of 800 L/h sterile air.