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A portable real-time fluorescence detection system was proposed for real-time detection of fresh pork freshness. The system consisted of two main parts, i.e., the research of nitrogen-doped graphene quantum dots (N-GQDs) fluorescent nanomaterials and the creation of portable fluorescence detection device. The morphology, structure and surface functional groups of N-GQDs were analyzed by transmission electron microscopy, Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, and it was confirmed that N-GQDs was successfully synthesized. The optical properties of N-GQDs were discussed, and it was proved that N-GQDs had excellent sensitivity and selectivity to amines. The response mechanism analysis showed that there was a static quenching effect between N-GQDs and NH3. The portable fluorescence sensor detection device included hardware design and software design, which mainly included five modules, namely light source excitation module, fluorescence detection module, temperature and humidity detection module, wireless communication module and threshold alarm module. Through the wireless communication module, the detection data was transmitted to the mobile phone APP and the host computer in real time, and the data was visualized and stored. A portable real-time fluorescence pork freshness detection system was developed by combining N-GQDs fluorescent nanomaterials with portable fluorescence detection device, and its performance was verified by experiments in fresh pork. The precise detection of TVB-N in pork at cold storage (4℃) and room temperature (25℃) showed excellent linear relationships (R225℃=0.97, R24℃=0.92).