The fabrication of bioactive scaffolds able to mimic the in vivo cellular microenvironment is a challenge for regenerative medicine. The creation of sites for the selective binding of specific endogenous proteins represents an attractive strategy to fabricate scaffolds able to elicit specific cell response. Here, electrospinning (ESP) and soft-molecular imprinting (soft-MI) techniques were combined to fabricate a soft-molecular imprinted electrospun bioactive scaffold (SMIES) for tissue regeneration. Scaffolds functionalized using different proteins and growth factors (GFs) arranged onto the surface were designed, fabricated and validated with different polyesters, demonstrating the versatility of the developed approach. The scaffolds bound selectively each of the different proteins used, indicating that the soft-MI method allowed fabricating high affinity binding sites on ESP fibers compared to non-imprinted ones. The imprinting of ESP fibers with several GFs resulted in a significant effect on cell behavior. FGF-2 imprinted SMIES promoted cell proliferation and metabolic activity. BMP-2 and TGF-β3 imprinted SMIES promoted cellular differentiation. These scaffolds hold the potential to be used in a cell-free approach to steer endogenous tissue regeneration in several regenerative medicine applications.