The emergence of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) necessitates novel therapeutics with distinct mechanisms. Here, we report amino-pyrazoline derivatives as a new class of dual-functional antimycobacterial agents, integrating potent bactericidal activity with fluorescence-based bacterial imaging. Initial screening identified AP-07 as a promising hit compound (MIC99: 40 μM against Mycobacterium smegmatis, 49 μM against Mycobacterium bovis BCG). Structure-based optimization led to the discovery of AP-02 and AP-05 as lead compounds, with enhanced activity (MIC99: 13-16 μM against M. smegmatis; 20-25 μM against M. bovis BCG). Additionally, spontaneous resistance assays detected no resistant colonies, suggesting a low risk of resistance development. Mechanistic studies confirmed Ag85C as the primary molecular target, disrupting late-stage mycolic acid biosynthesis and impairing cell wall integrity. Notably, pyrazoline derivatives exhibit intrinsic fluorescence, selectively labeling intracellular mycobacteria while remaining non-toxic to host macrophages, enabling real-time bacterial imaging. This work establishes fluorescent amino-pyrazolines as a promising foundation for next-generation antitubercular agents, bridging diagnostics and therapy in tuberculosis drug discovery.