Objective To construct a system for studying the ultrastructure and mechanical properties of insect flight muscle fiber in different activated states so as to carry out cardiac biomechanics study in physiological environment, and further promote understanding of the relationship between cardiac structure, mechanical properties and physiological function, and provide more clues for the basic and clinical research on cardiac diseases. Methods The ultrastructure of insect flight muscle fibrils in rigor, relaxed and activated state was investigated using the tapping mode of atomic force microscopy (AFM), and the elasticity of muscle fibers in different physiological states was studied using the nanoindentation. Results Sarcomere lengths of insect flight muscle fiber in rigor, relaxed and activated state were (2.10±0.05), (3.10±0.10), (2.50±0.15) μm (2 mmol/L Ca2+), (2.60±0.25) μm (5 mmol/L Ca2+) and (2.55±0.15) μm (10 mmol/L Ca2+), respectively, while the A-band length maintained at 1.50 μm and I-band changed from 0.7~1.6 μm. Mechanical test found that the elasticity of different bands or lines in the same physiological state varied in the order of Z-line>M-line>overlap>I-band. Conclusions Critical Ca2+ concentration for muscle fiber activation was 5 mmol/L, and sarcomere length distributions were in line with the relative slip theory and structure model, and AFM was the potential tool for the high resolution study on ultrastructure and mechanical properties of the muscle fibers.