Owing to the increase in the global demand of meat, cultured meat technology is being developed to circumvent a shortage of meat in the future. However, methods for construction of millimetre-thick bovine muscle tissues with highly aligned myotubes have not yet been established. Here, we propose a culture method for constructing 3D-cultured bovine muscle tissue containing myotubes aligned along its long-axial direction, which contracted in response to electrical stimulation. First, we optimised the composition of biomaterials used in the construction and the electrical stimulation applied to the tissue during culture. Subsequently, we fabricated millimetre-thick bovine muscle tissues containing highly aligned myotubes by accumulating bovine myoblast-laden hydrogel modules. The microbial content of the bovine muscle tissue cultured for 14 days was below the detection limit, indicating that the muscle tissues were sterile, unlike commercial meat. Therefore, the proposed construction method for bovine muscle tissues will be useful for the production of clean cultured steak meat simulating real meat.