The uncontrolled overuse of tetracyclines (TCs) has contributed to a significant rise in the levels of not only antibiotic residues in the environmental but also drug- and multidrug-resistant bacteria, necessitating a convenient, sensitive, and rapid assay for the quantitative determination and health risk assessment of TCs. This study presents the development of a nanocomposite sensor which integrates hydroxylated multiwalled carbon nanotubes (MWCNTs-OH), tungsten disulfide quantum dots (WS2QDs), and prussian blue (PB). This sensor was capable of measuring the concentrations of tetracycline (TC), oxytetracycline (OTC), doxycycline (DC), and tigecycline (TEC) at levels ranging from 0.10 to 125.00 μM, with detection limits of 0.010, 0.015, 0.020, and 0.060 μM, respectively (S/N = 3). The excellent electrochemical performance enabled the sensor to measure TC concentrations in real water samples. Importantly, the sensor was also used to evaluate the cytotoxicity of TCs on human hepatocarcinoma (HepG2) cells. The 48 h half-inhibitory concentrations (IC50) for TC, OTC, DC, and TEC were 344.91 μM, 365.14 μM, 349.59 μM, and 604.66 μM, respectively, indicating that TC was the most cytotoxic. The sensing platform described herein not only facilitates the quantitative determination of TCs but also allows for the assessment of their cytotoxicity in the water environment, which greatly simplifies the comprehensive evaluation process and reduces the cost, thus holding great promise for TCs testing.