Muscle injuries present a significant challenge in sports and physical activity, necessitating advanced diagnostic tools. Current invasive methods, such as muscle biopsies, are limited by procedural risks and lack of dynamic data. In this study, we developed a dual-channel fluorescent biosensor based on MoS2 nanosheets and fluorescence resonance energy transfer (FRET) for the simultaneous detection of miR-133a and ATP in living mouse myoblast cells. The design leverages the pathophysiological roles of miR-133a (a biomarker upregulated during muscle injury) and ATP (a critical indicator of energy metabolism). The biosensor incorporates FAM-labeled miR-133a-specific ssDNA and Cy5-labeled ATP aptamer probes immobilized on MoS2 nanosheets, achieving fluorescence quenching via FRET. Target binding triggered fluorescence recovery, enabling sensitive detection with limits of detection (LOD) of 3.3 pM for miR-133a and 0.33 µM for ATP. In vitro experiments confirmed high specificity and biocompatibility in mouse myoblasts, validated through confocal microscopy and flow cytometry. This platform provides a foundational tool for monitoring molecular dynamics associated with muscle injury, focusing on miR-133a and ATP levels in cellular environments. This work addresses limitations of traditional invasive diagnostics and lays the groundwork for potential applications in sports medicine research.