Aldo-keto reductases (AKRs) are widely used for reducing prochiral aldehyde or ketone compounds into corresponding chiral alcohols. In our previous work, an aldo-keto reductase named YtbE was isolated and identified from Bacillus sp. ECU0013, which can catalyze an NADPH-dependent carbonyl reduction reaction with high stereoselectivity. In order to further expand the application range of the enzyme, three-Dimensional Quantitative Structure Activity Relationship (3D-QSAR) models were constructed to predict the specific activity on the aldehyde or ketone compounds. For Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Indices Analysis (CoMSIA) models, both the cross-validation coefficient (Q²) (0.623, 0.601) are satisfied, suggesting that the models are robust and effective for prediction. The detailed structure-function relationships between the protein YtbE and different substrates were then examined by the contour maps analysis. We found that electrostatic and hydrophobic force molecular fields play the most important roles in determining the catalytic activity, with contribution factors of 0.378 and 0.384, respectively. Furthermore, virtual screening by using the ZINC15 database was performed to expand investigate the substrate spectrum of YtbE. Among the 77 potential substrates predicted, five were experimentally verified, indicating the better accuracy of CoMSIA-molecule model. The 3D-QSAR assisted method was proved to be helpful for rational investigating and expanding of the YtbE substrate spectrum.