Stem cell transplantation is considered promising for treating cardiovascular disorders. However, lack of an effective carrier impacts their survival and proliferation. Poly 3-hydroxybutyrate-co-4-hydroxybutyrate [P(3HB-co-4HB)] is a novel polymer biomaterial with excellent physical characteristics. We assessed the suitability of P(3HB-co-4HB) combined with embryonic stem cells (ESCs) for myocardial patch formation. Mouse ESC embryoid bodies (EBs) were induced into cardiomyocytes in the absence (control group) and presence of 0.1 mg/ml vitamin C (vitamin C group). The myocardial patch was observed by confocal and scanning electron microscopy. Staining of cardiomyocyte-specific markers, cTnT and a-actin, was carried out from day 7 of EB formation throughout patch co-culture every two days. Adherent mouse ESCs displayed normal growth and morphology. When cultured as EBs for 3 days, 100% grew on the biofilm and after 7 days cells were present in the 3D biofilm to a depth of 2 mm, with normal cardiomyocyte morphology. Cardiomyocytes were cTnT- and a-actin-positive. EB morphology in both groups was normal, however the differentiation efficiency of the vitamin C group was significantly higher than the control group (p = 0.026, P < 0.05). Furthermore, the induction rates of the control and vitamin C group were 17.8% and 71.1%, respectively. P(3HB-co-4HB) permitted ESC growth and differentiation forming a myocardial patch. Adding vitamin C significantly increased the induction rate of ESCs into cardiomyocytes. P(3HB-co-4HB) is a suitable biomaterial for investigating cardiomyocyte regeneration in patients with myocardial infarction.