The bioreduction of prochiral ketones offers efficient access to chiral secondary alcohols, which are potentially beneficial precursors for producing many biologically active compounds and natural products. This bioreduction process can be affected by different parameters when whole-cell of biocatalysts such as Lactic Acid Bacteria strains are applied. In this context, this paper proposed an I-optimal design-based model to optimize culture parameters such as temperature, pH, incubation period, and agitation speed for asymmetric bioreduction of 2-methyl-1-phenylpropan-1-one (1) with Lactobacillus fermentum BY35 as a biocatalyst while achieving the highest conversion rate (cr) and enantiomeric excess (ee). The optimum settings of the four culture parameters and the cr and ee values were found using the proposed optimization model as follows: pH = 6.5, temperature = 25 degrees C, incubation period = 38.5 h, agitation speed = 200 rpm, the ee value = 98.78%, and the cr value = 98.92%. After the validation of the process, the cr and ee values were found to be > 99% and > 99%, respectively, while using the optimum operating conditions from the optimization model. Thus, the results of the optimization model are consistent with the results of the validation experiment. It is also noted that this paper is the first to optimize culture parameters using the proposed I-optimal design-based model for an asymmetric reduction.