Doxorubicin (DOX) is widely used in chemotherapy as an anti-tumor drug. However, DOX is highly cardio-, neuro- and cytotoxic. For this reason, the continuous monitoring of DOX concentrations in biofluids and tissues is important. Most methods for the determination of DOX concentrations are complex and costly, and are designed to determine pure DOX. The purpose of this work is to demonstrate the capabilities of analytical nanosensors based on the quenching of the fluorescence of alloyed CdZnSeS/ZnS quantum dots (QDs) for operative DOX detection. To maximize the nanosensor quenching efficiency, the spectral features of QDs and DOX were carefully studied, and the complex nature of QD fluorescence quenching in the presence of DOX was shown. Using optimized conditions, turn-off fluorescence nanosensors for direct DOX determination in undiluted human plasma were developed. A DOX concentration of 0.5 µM in plasma was reflected in a decrease in the fluorescence intensity of QDs, stabilized with thioglycolic and 3-mercaptopropionic acids, for 5.8 and 4.4 %, respectively. The calculated Limit of Detection values were 0.08 and 0.03 μg/mL using QDs, stabilized with thioglycolic and 3-mercaptopropionic acids, respectively.