Obesity is a growing global health concern, contributing to various metabolic disorders. Despite progress in anti-obesity research, existing in vitro cell culture has some limitations, which fail to have extracellular matrix (ECM) and microenvironment of adipose tissue, while To address these challenges, novel 3D cultures and microfluidic models are being developed. Here, we developed a 3D adipose tissue-on-a-chip model to replicate in vivo adipogenesis, focusing on ECM strength, which is crucial for adipocyte differentiation and function but often underestimated. Using polydimethylsiloxane (PDMS), we created a microfluidic chip with embedded microchannels for controlled medium flow. We optimized the size of 3T3-L1 preadipocyte spheroids and cultured them in hydrogel environments with different concentrations of gelatin methacryloyl (GelMA) (3%, 5%, and 10%) to identify optimal ECM conditions for adipogenesis. Our results show that 3% GelMA hydrogel promoted the highest spheroid maturation, as evidenced by increased lipid droplet formation, spheroid diameter, and enhanced expression of adipogenesis-related genes, including PPARγ and C/EBPα. This suggests that ECM strength plays a key role in adipocyte differentiation. This adipose-on-a-chip model offers a promising in vitro platform for studying adipose tissue function and obesity-related pathologies, with potential applications in anti-obesity therapy testing and disease modeling.