Current approaches to dealing with the worldwide problem of marine biofouling are to impart chemical functionality to the surface or utilize microtopography inspired by nature. Previous reports have shown that only introducing a single method may not resist adhesion of mussels or inhibit biofouling in static forms. While it is promising to integrate two methods to develop an effective antifouling strategy, related basic research is still lacking. Here, we have fabricated engineered shark skin surfaces with different feature heights and terminated with different chemical moieties. Atomic force microscopy (AFM) with a modified colloid probe technique and quartz crystal microbalance with a dissipation n (QCM-D) monitoring method have been introduced to directly determine the interactions between adhesive proteins and functionalized surfaces. Our results indicate that the adhesion strength of probe-surface decreases with increasing feature height, and it also decreases from bare Si surface to alkyl and hydroxyl modification, which is attributed to different contact area domains and interaction mechanisms. Combining biomimetic microtopography and surface chemistry, our study provides a new perspective for designing and developing underwater anti-fouling materials.