In nature, many biological surfaces exhibit inherent bacteriostasis due to the existence of special microstructures. However, the key factors and underlying mechanisms driving this property remain unclear. A significant challenge lies in the lack of proper techniques for precisely fabricating such microstructures as well as finely tuning their morphological parameters. In this study, we adopted a two-photon 3D printing-based approach to fabricate microstructures on specified surfaces with accurate control over their morphology, enabling investigation on structural bacteriostasis. Through abstracting the subtle morphology on shark skin, we replicated their bacteriostatic microstructures and were able to regulate their morphology at the micron scale. By culturing Streptococcus mutans on the surface of these microstructures, we validated their bacteriostatic performance and demonstrated that morphological parameters significantly influenced the efficacy of structural bacteriostasis. Other kinds of microstructures such as micro-holes and their bacteriostasis could also be fabricated and investigated utilizing this two-photon polymerization technology. We believe this strategy offers a powerful tool for researching bacteriostatic mechanisms of various microstructures and will inspire their broad applications in both daily and industrial settings.