Progress in cardiac cell replacement therapies and tissue engineering critically depends on our ability to isolate functional cardiomyocytes (CMs) from heterogeneous cell mixtures. Label-free enrichment of cardiomyocytes is desirable for future clinical application of cell based products. Taking advantage of the physical properties of CMs, a microfluidic system was designed to separate CMs from neonatal rat heart tissue digest based on size using the principles of deterministic lateral displacement (DLD). For the first time, we demonstrate enrichment of functional CMs up to 91 ± 2.4% directly from the digested heart tissue without any pre-treatment or labeling. Enriched cardiomyocytes remained viable after sorting and formed contractile cardiac patches in 3-dimensional culture. The broad significance of this work lies in demonstrating functional cell enrichment from the primary tissue digest leading directly to the creation of the engineered tissue.