Structural similarity of electrospun fibers (ESFs) to the native extracellular matrix provides great potential for the application of biofunctional ESFs in tissue engineering. This study aimed to synthesize biofunctionalized poly (Llactide-co-glycolide) (PLGA) ESFs for investigating the potential for cardiac tissue engineering application. We developed a simple but novel strategy to incorporate adhesive peptides in PLGA ESFs. Two adhesive peptides derived from laminin, YIGSR, and RGD, were covalently conjugated to poly-L-lysine, and then mingled with PLGA solution for electrospinning. Peptides were uniformly distributed on the surface and in the interior of ESFs. PLGA ESFs incorporated with YIGSR or RGD or adsorbed with laminin significantly enhanced the adhesion of cardiomyocytes isolated from neonatal rats. Furthermore, the cells were found to adhere better on ESFs compared with flat substrates after 7 days of culture. Immunofluorescent staining of F-actin, vinculin, a-actinin, and Ncadherin indicated that cardiomyocytes adhered and formed striated a-actinin better on the laminin-coated ESFs and the YIGSR-incorporated ESFs compared with the RGD-incorporated ESFs. The expression of a-myosin heavy chain and b-tubulin on the YIGSR-incorporated ESFs was significantly higher compared with the expression level on PLGA and RGD-incorporated samples. Furthermore, the contraction of cardiomyocytes was faster and lasted longer on the laminin-coated ESFs and YIGSR-incorporated ESFs. The results suggest that aligned YIGSRincorporated PLGA ESFs is a better candidate for the formation of cardiac patches. This study demonstrated the potential of using peptide-incorporated ESFs as designable-scaffold platform for tissue engineering.