A novel optical nano-sensor for tetracycline hydrochloride (TH) detection as a small molecule model was prepared by modifying the surface of CdTe/ZnS QDs with a biopolymer based cap, called poly ((2-dimethylaminoethyl) methacrylate) grafted onto oxidized starch (OS-g- PDMAEMA), via a facile method. The preparation of the nano-sensor was optimized by employing a 4-factor Box–Behnken statistical design to achieve a nano-sensor with maximum fluorescence intensity for sensitive determination of TH in real samples. The optimized nano-sensor based on CdTe/ZnS QDs were characterized by fluorescence spectroscopy, UV–vis spectrophotometry, transmission electron microscopy (TEM), Fourier transforms infrared (FTIR) and thermo-gravimetric (TG) analysis techniques. It was found that the fluorescence intensity of modified QDS (CdTe/ZnS-OS-g-PDMAEMA QDs) at 520 nm (excitation at 375 nm) was selectively quenched in the presence of trace amounts of TH drug. In this method, a simple, highly sensitive, selective and rapid analytical approach was used for the determination of TH in the concentration range of 9.14 × 10−9 mol L−1–7.23 × 10−6 mol L−1 with a detection limit of 2.74 × 10−9 mol L−1. The designed nano-sensor can be successfully applied for the determination of TH in complex matrixes (pharmaceutical formulations, urine, saline and glucose serums) with satisfactory analytical results.