β-Lactoglobulin amyloid fibril (BLGF)-capped gold nanoclusters (Au NCs) with red, green and blue emissions were fabricated via pH-dependent reduction strategy. The BLGF-Au NCs exhibited 3.2 times enhancement of fluorescence (λex = 500 nm, λem = 684 nm), a significant 42 nm red shift, a 11.57% quantum yield and a 1.4 μs decay time compared with native β-lactoglobulin (BLG)-stabilized Au NCs. Meanwhile, the multicolor Au NCs were employed for cell imaging via incubation with A549 cells for 14 h. According to the Michaelis-Menten equation, the kinetic parameters of the BLGF-Au NCs showed a lower Km value (66 μmol L-1) for 3,3,5,5-tetramethylbenzidine (TMB) and a higher vmax (3.74 × 10-8 M s-1) for H2O2, which are comparable with other artificial nanoenzymes and natural peroxidases. Based on the highly intrinsic peroxidase-like activity of the BLGF-Au NCs, a colorimetric method was developed for glucose determination with a detection limit of 1.5 μmol L-1 by determining the variation of the absorption at 652 nm, ranging from 5 to 100 μmol L-1. In addition, the glucose assay method also revealed a 101.02 to 104.16% recovery in a real human serum sample.