Convenient, rapid and reliable detection of food spoilage microorganisms has always been critical to food safety. Commonly used methods such as an enzyme-linked immunosorbent assay (ELISA) often rely on tedious wash steps and incubation procedures. A convenient two-step colorimetric homogeneous immunoassay strategy for rapid visual detection of Zygosaccharomyces rouxii, which efficiently simplified the immunoassay process, was developed. Immunogold nanospheres (Ab–PEG–AuNPs) were used as specific colorimetric probes. The proposed method depends on adjusting the interparticle spacing of Ab–PEG–AuNPs on the cell surface of Z. rouxii, leading to a visible color change. This simple method exhibits good sensitivity and specificity which depends on the characteristic of Ab against Z. rouxii, and the detection results are in great agreement with those of ELISA. Moreover, the method is successfully applied to detect Z. rouxii in apple juice.