Rapid and simple monitoring of vancomycin (VAN) concentration in blood is a vital strategy for maximizing therapeutic efficacy, minimizing toxicity and developing a personalized treatment plan. In this work, a simple multicolor immunosensor is proposed to enable rapid monitoring of VAN concentration in serum, without using any expensive and bulky instrument. The multicolor immunosensor platform is a system that works based on the principle that the product of cetyltrimethylammonium bromide-blue oxide of 3,3',5,5'-tetramethylbenzidine interaction (CTAB/TMB+) and TMB+ increases simultaneously with the decrease in VAN concentration, whereas AuNBPs are insensitive to VAN. The result indicates that the reaction system has multiple distinct color variants. These distinct vivid color changes can be easily distinguished with the naked eye, and smartphone-relied red-green-blue (RGB) analysis can be used for quantitative detection, without the need for any sophisticated apparatus. The construction of this multicolor system omitted the hydrochloric acid (HCl) addition step, growth or etch procedure of noble metal nanoparticles in traditional multicolor immunosensors, which can improve the time-cost and tedious operation. The proposed method achieves a good linear relationship (r2 = 0.9679), accuracy (recoveries, 99.25-126.96%) and repeatability (n = 3, RSD, 1.27-2.17%). Moreover, a good correlation was observed between the results obtained from the new method and liquid chromatography-tandem mass spectrometry (r2 = 0.8993, n = 8). In summary, this work provides a new low-cost, facile and user-friendly immunosensor platform with high potential for rapid detection of VAN and various other drugs at home, hospital rooms and rural areas.