In tissue engineering, autofluorescence of polymer scaffolds often lowers the image contrast, making it difficult to examine cells and subcellular structures. Treating the scaffold materials with Sudan Black B (SBB) after cell fixation can effectively suppress autofluorescence, but this approach is not conducive to live cell imaging. Post-culture SBB treatment also disrupts intracellular structures and leads to reduced fluorescence intensity of the targets of interest. In this study, we introduce pre-culture SBB treatment to suppress autofluorescence, where SBB is applied to polymeric scaffold materials before cell seeding. The results show that the autofluorescence signals emitted from polycaprolactone (PCL) scaffolds in three commonly used fluorescence channels effectively decrease without diminishing the fluorescence signals emitted from the cells. The pre-culture SBB treatment does not significantly affect cell viability. The autofluorescence suppressive effect does not substantially diminish during the culturing period up to 28 days. The results also show that cell migration, proliferation, and myogenic differentiation in pre-culture SBB-treated groups do not exhibit statistical difference from the non-treated groups. As such, this approach greatly improves the fluorescence image quality for examining live cell behaviors and dynamics while the cells are cultured within autofluorescent polymer scaffolds.