Streptozocin (STZ) is a broad range antibiotic, highly genotoxic, antineoplastic and hyperglycemic. HSA is the most abundant protein in physiology and it binds to almost all exogenic and endogenic ligands, including drugs. STZ-induced fluorescence quenching of HSA has been done at pH 7.4, pH 3.5 and at pH 7.4 with 4.5 M urea at temperatures 286 K, 291 K, and 306 K. K_sv found to be 10³ M⁻¹, binding constant 1.5X10³M⁻¹ and binding sites ~1. But, K_sv for HSA and glucopyranose interaction was found lesser than that of HSA-STZ binding. Binding of STZ/glucopyranose on HSA seems to result in complex formation as calculated K_q > 10¹⁰ M⁻¹ s⁻¹. The number of binding sites, binding constants, and binding energies were increased with temperature. The ΔG⁰, ΔH⁰, and ΔS⁰ for HSA-STZ interaction were found to be −17.7 × 10³ J·mol⁻¹; 2.34 × 10⁵ J·mol⁻¹ and 841 JK⁻¹ mol⁻¹ respectively at pH 7.4 and 291 K. The comparative bindings of N, F and I states of HSA with STZ and their molecular docking analyses indicate that IIIA-B junction (i.e., inter-helix h6_DOM3-h7_DOM3) is the probable binding site, a locus close to fatty acid binding site-5. These results could be useful for therapeutic and analytical exploitation of STZ, as albumin used as the vehicle for drug delivery.