In the presented work, simple, green, sensitive, and selective nitrogen-doped carbon quantum dots (N-CQDs) were developed as nano-sensor for quantification of tigecycline (TIG) in different matrices. The proposed method is based on microwave synthesis of green nitrogen-doped carbon quantum dots with a high quantum yield (41.39%) and size diameter equal to 2.0 nm from the green juice of Eruca sativa leaves. The relative fluorescence intensity (RFI) of the green synthesized quantum dots (N-CQDs) was quenched at emission 512 nm (excitation 445 nm) after the addition of TIG drug. A good linear range between TIG concentration and quenched fluorescence intensity of N-CQDs in the range 20–300 ng mL−1, with the lower limit of quantitation (LOQ) equal to 8.51 ng mL−1. The proposed method was validated using the international conference of harmonization (ICH) recommendation and bio-analytical validation using U.S. food and drug administration (US-FDA) guidelines. The N-CQDs have been fully characterized using a transmission electron microscope (TEM), dynamic light scattering (DLS), X-Ray photoelectron spectroscopy (XPS), and Fourier-transform infrared spectrometer (FTIR). The suggested technique is a straightforward analytical procedure that can be used in clinical laboratories. Under the optimum condition, TIG was estimated in human plasma with a high percentage of recovery ranging from 96.95 to 98.54%. In addition, the proposed method was applied effectively in milk samples with percentage of recovery equal to 98.90 ± 1.55.