Alkane hydroxylase (AlkB), a membrane-bound enzyme has high industrial demand; however, its economical production remains challenging due to its intrinsic nature and co-factor dependency. In the current study, various critical process parameters for optimum production of AlkB have been optimized through feed forward neural network (FFNN) and genetic algorithm (GA) models using Penicillium chrysogenum SNP5 (MTCC13144). AlkB specific activity under preliminary un-optimized conditions i.e., 1% hexadecane, 7.4 pH, 11 days incubation time, 28 °C incubation temperature and 1 ml of inoculum size was 100 U/mg. ‘One variable at a time’ (OVAT) strategy was used to identify optimum physicochemical parameters and then its output data was fed to develop a model of FFNN with ‘6-12-1’ topology. Outputs of FFNN were further optimized through GA to minimize errors and intensify search level. This has provided superior predictive performances with 0.053 U/mg overall mean absolute percentage error (MAPE), 6.801 U/mg root mean square errors (RMSE), and 0.987 overall correlation coefficient (R). The AlkB specific activity improved by 3.5-fold, i.e., from 100 U/mg under preliminary un-optimized conditions to 351.32 U/mg under optimum physicochemical conditions obtained through FFNN-GA hybrid method, i.e., hexadecane (carbon source): 1.56% v/v, FeSO4: 0.63 mM, incubation temperature: 27.40 °C, pH: 7.38, incubation time: 12.35 days and inoculums size: 1.33 ml. The developed process would be a stepping stone to fulfill the high industrial demands of  Alkane hydroxylase.