A fluorescence analysis method based on gold nanocluster (AuNC) and metal–organic framework (MOF) composite materials (AuNCs@ZIF-8) was established for highly sensitive detection of bilirubin (BR). First, AuNCs@ZIF-8 was successfully obtained by co-precipitation and displayed an aggregation-induced emission enhancement by the confinement effect of the MOFs (i.e., ZIF-8). The product showed approximately 7.0 times enhancement in the quantum yield and longer fluorescence lifetime from 2.29 μs to 11.51 μs compared with AuNCs. When BR combined with the metal node Zn²⁺ of ZIF-8, the skeleton of the composite was destroyed, leading to a great decrease in the fluorescence intensity by the transformation of the AuNCs from the aggregated state to dispersed state. The linear range for the detection of BR was 0.1–5.0 μM, with the limit of detection (LOD) of 0.07 μM (S/N = 3). The AuNCs@ZIF-8 exhibited a selective response toward BR within 5 min and detected BR in human serum. The long-wavelength emission by AuNCs avoided the interference of the complex biomatrix background fluorescence, indicating their great application prospects for clinical diagnosis.