Cardiovascular mortality remains a major health challenge. Cardiomyocyte (CM)-based tissue engineering (TE) offers promising alternatives for developing therapies via in vitro models. However, the immature phenotype of CM in engineered tissues hampers progress. Recent studies introduce conductive materials like graphene to enhance CM maturation, but conventional graphene synthesis suffers from complexity, toxicity, and low yield. Laser-induced graphene (LIG) provides a sustainable, cost-effective, eco-friendly solution with efficient conductivity and biocompatibility. A LIG-based substrate is bioengineered in this study, hypothesizing that its conductive, anisotropic properties promote CM maturation and mimic the native cardiac niche. LIG is fabricated using a CO2 laser with Parylene-C as a precursor. Stem cells (SCs) and SC-derived embryoid bodies (EBs) are cultured on LIG substrates, and their viability, metabolic activity, morphology, and protein expression are evaluated through immunofluorescence and electron microscopy. Both SCs and EBs maintain viability and activity throughout the culture. Moreover, EB-derived CM exhibit spontaneous contraction and express cardiac-specific proteins, confirming functional differentiation on LIG matrices. This first report demonstrates that LIG substrates support SC culture and differentiation, highlighting their potential in developing refined in vitro cardiac models and advancing regenerative therapeutic strategies. The findings support LIG as a transformative advancement in TE.