Organ-on-a-chip stands as a pivotal platform for skeletal muscle research while constructing 3D skeletal muscle tissues that possess both macroscopic and microscopic structures remains a considerable challenge. This study draws inspiration from LEGO-like assembly, employing a modular approach to construct muscle tissue that integrates biomimetic macroscopic and microscopic structures. Modular LEGO-like hybrid nanofibrous scaffold bricks were fabricated by the combination of 3D printing and electrospinning techniques. Skeletal muscle cells cultured on these modular scaffold bricks exhibited a highly orientated nanofibrous structure. A variety of construction of skeletal muscle tissues further enabled development by various assembling processes. Moreover, skeletal muscle-on-a-chip (SMoC) was further assembled as a functional platform for electrical or perfusion stimuli investigation. The electrical stimulus was conveniently applied and tuned in such a SMoC platform to significantly enhance the differentiation of skeletal muscle tissues. Additionally, the effect of perfusion stimulation on skeletal muscle vascularization within the SMoC platform was also demonstrated. These findings highlight the potential of these assembled SMoCs as functional ex vivo platforms for skeletal tissue engineering and drug research applications, and such a LEGO-like assembly strategy could also be applied to the other engineering organ-on-chips fabrication, which facilitates the development of bionic functional platforms for various biomedical research applications. We developed a list of modular nanofibrous scaffold bricks by a hybrid fabrication method combining 3D printing and electrospinning techniques, featuring precise microscale and nanoscale structures. Emulating the LEGO-like assembly method, these bricks were assembled along the x–y–z axis to mimic various skeletal muscle structures. These developed engineered skeletal muscle tissues were further integrated into the microfluidic chip to develop the skeletal muscle-on-a-chip (SMoC) as an in vitro testing platform for both electrical and perfusion stimuli investigation.