Levamisole is a broad-spectrum anti-insect drug widely used to fight against nematodes in pigs, cattle and other livestock. Meanwhile, levamisole has a special immunomodulatory effect often used in anti-bacterial, anti-inflammatory, anti-virus and growth-promoting aspects in animal breeding. When it is used irrationally, it is easy to produce residues in poultry meat. At present, the common detection methods of levamisole are liquid chromatography and gas chromatography, which have the disadvantages of complex operation, long time consumption and high cost. Surface-enhanced Raman spectroscopy (SERS) has the advantages of fast analysis speed, high detection sensitivity and good specificity and has been widely used in the rapid detection of pesticide residues and animal residues in recent years. In order to realize the rapid detection of levamisole residues in pork, a rapid detection method of levamisole residues in pork by SERS was established. Through a single factor experiment, the optimal volume ratio of gold colloid to sample solution and the optimal integration time are determined to be 2∶1 and 20 s, respectively. By comparing the extraction effects of different extraction methods and solvents on the residual levamisole hydrochloride in pork, the extraction conditions of centrifugation after liquid-liquid extraction with n-hexane and redissolution with nitrogen blowing were determined. The theoretical spectrum of levamisole hydrochloride was calculated by B3LYP/6-311+G(d) basis set in density functional theory. After optimizing the molecular structure, the frequency and Raman spectrum was calculated. The theoretically calculated spectrum was in good agreement with the peaks of the solid spectrum and solution spectrum. According to theoretical calculation spectrum, solid spectrum and solution spectrum, SERS characteristic peaks of levamisole hydrochloride are determined, and vibration attribution is carried out. The characteristic peaks at 469, 627 and 969 cm^-1 are obtained as characteristic quantitative peaks of levamisole hydrochloride, in which 469 cm^-1 is C—S bond stretching vibration, 627 cm^-1 is benzene ring C—C bending deformation vibration, and 969 cm^-1 is imidazole ring in-plane bending and side chain skeleton vibration. Under the optimum experimental conditions, the standard curve of SERS signal and levamisole hydrochloride standard solution concentration was established, and the R² values of linear equations were all above 0.9. The average recovery of levamisole hydrochloride with different spiked concentrations was 80.39%~95.94%, and RSD was 3.08%~6.20%. The method is stable and straight forward and can be used for rapid and accurate determination of levamisole residues in pork without complex pretreatment.