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Herein, the boron and nitrogen co-doped 0-dimensional graphene quantum dots (B,N-GQDs) with high quantum yield (QY) were synthesized via microwave-assisted hydrothermal method at 170 °C for 20 min using fresh passion fruit juice and boric acid as the starting materials. The 3–6 layers of B,N-GQDs with mean particle size of 9 ± 1 nm were then used for ultra-sensitive and selective detection of tetracycline in aqueous and biological media. The hybridization of boron and nitrogen atoms into the GQD structures increases the intensity of electronegative, resulting in the enhancement of QY to 50 ± 1%. The B,N-GQDs show their excellent analytical performance on tetracycline determination after 2 min of reaction under an optimal condition at pH 5. The linear range of 0.04–70 µM and with limits of detection (LOD) of 1 nM in phosphate buffer saline (PBS), 1.9 nM in urine and 2.2 nM in human serum are obtained. Moreover, the high selectivity of tetracycline by B,N-GQDs over the other 23 interferences is observed. The π-π interaction and electron donor-acceptor principle play pivotal roles in enhancing the ultra-sensitivity and selectivity of B,N-GQDs toward TC detection. Moreover, the B, N-GQD based paper nanosensor exhibits an excellent analytical performance on visual detection of 0.1–30 µM TC in human serum. Results of this study clearly indicate the feasibility of synthesis of B,N-GQDs derived from passion fruit juice for ultrasensitive tetracycline detection, which can open an avenue to use natural products for the preparation of environmentally benign and biocompatible carbon nanomaterials for highly sensitive detection of drugs, antibiotics, organic compounds and biomarkers.