The fabrication of iron oxide quantum dots (IO-QDs) modified with glutamic acid (Glu) under controllable conditions is reported. The IO-QDs have been characterized by transmission electron microscopy, spectrofluorometry, powder X-ray diffraction, vibrating sample magnetometry, UV-Vis spectroscopy, X-ray photoelectron spectroscopy, and Fourier-transform infrared spectroscopy. The IO-QDs exhibited good stability towards irradiation, temperature elevations, and ionic strength, and the quantum yield (QY) of IO-QDs was calculated to be 11.91 ± 0.09%. The IO-QDs were furtherly measured at an excitation wavelength of 330 nm with emission maxima at 402 nm, which were employed to detect tetracycline (TCy) antibiotics, including tetracycline (TCy), chlortetracycline (CTCy), demeclocycline (DmCy), and oxytetracycline (OTCy) in biological samples. The results indicated that TCy, CTCy, DmCy, and OTCy in urine samples show a dynamic working range between 0.01 and 80.0 μM; 0.01 and 1.0 μM; 0.01 and 10 μM; and 0.04 and 1.0 μM, respectively, with detection limits of 7.69 nM, 120.23 nM, 18.20 nM, and 67.74 nM, respectively. The detection was not interfered with by the auto-fluorescence from the matrices. In addition, the obtained recovery in real urine samples suggested that the developed method could be used in practical applications. Therefore, the current study has prospect to develop an easy, fast, eco-friendly, and efficient new sensing method for detecting tetracycline antibiotics in biological samples.