The aims of the this study are to select the best cultivation type for plant growth regulator (PGR) production, to optimize PGR production with statistical experimental design, and to calculate bioprocess parameters and yield factors during PGR production by P. eryngii in flask and reactor scales. Submerged fermentation was the best cultivation type with 4438.67 ± 37.14, 436.95 ± 27.31, and 54.32 ± 3.21 mg/L of GA3, ABA, and IAA production values, respectively. The Plackett–Burman and Box–Behnken designs were used to determine effective culture parameters and interactive effects of the selected culture parameters on PGR production by Pleurotus eryngii under submerged fermentation. The statistical model is valid for predicting PGR production by P. eryngii. After these studies, maximum PGR production (7926.17 ± 334.09, 634.92 ± 12.15, and 55.41 ± 4.38 mg/L for GA3, ABA, and IAA, respectively) was reached on the 18th day of fermentation under optimized conditions. The optimum formula was 50 g/L fructose, 3 g/L NaNO3, and 1.5 g/L KH2PO4, 1 mg/L thiamine, incubation temperature 25 °C, initial medium pH 7.0, and an agitation speed of 150 rpm. The kinetics of PGR production was investigated in batch cultivation under 3-L stirred tank reactor conditions. Concentrations of GA3, ABA, and IAA of 10,545.00 ± 527.25, 872.32 ± 21.81, and 60.48 ± 3.48 mg/L were obtained at the reactor scale which were 4.1, 3.4, and 2.3 times higher than the initial screening values. The specific growth rate (µ), the volumetric (r p) and specific (Q p) PGR production rates, 486.11 mg/L/day and 107.43 mg/g biomass/day for GA3, confirmed the successful transfer of optimized conditions to the reactor scale. In the presented study, PGR production of P. eryngii is reported for the first time.