CdTe quantum dots (QD-CdTe) functionalized with mercaptosuccinic acid (MSA) were synthesized in an aqueous medium, varying synthesis time from 0.5 to 4 h. The nanoprobe were characterized by a direct relationship between synthesis time and QD size (2.61-3.04 nm). The QD-CdTe-MSA interacted with protamine (PT), a cationic protein, forming a bioconjugate, thus quenching the photoluminescence intensity and generating an on-off system. The nanoprobe produced at a synthesis time of 1 h (QD-CdTe1) presented PT’s best sensitivity in a succinate buffer (pH = 5). Under the optimized conditions, the proposed method presented a linear range of 0.05-0.5 mg L-1 (10-100 nM), limit of detection (LOD) 0.01 mg L-1 (2 nM), and relative standard deviation (RSD) ≤ 2.01% (n = 10). The interaction of the nanoprobe and PT led to aggregation due to a bioconjugate formation. The systems’ hydrodynamic radius varied from 4.31 nm (QD‑CdTe1) to 30.50 nm for the bioconjugate (QD-CdTe1-PT). The method was sensitive to variation in ionic strength and based on thermodynamic parameters; it was demonstrated that the interaction mechanism occurred preferentially through electrostatic forces. Finally, the method proved to be fast, sensitive, and viable for quantifying PT in drugs and synthetic urine samples with recoveries above 95%.