In this paper, a high-yield, monodisperse Au nanobipyramids (Au NBs) sol was prepared by seed-mediated method, and gold nanoparticles were assembled on the surface of the silicon wafer by self-assembly technology to obtain a solid SERS substrate. Scanning electron microscopy (SEM) showed that the average length of Au NBs was 34.31 nm, and the analysis enhancement factor (AEF) was approximately 7.3×10 5 with rhodamine 6G (R6G) as a probe. SERS detection of chlortetracycline hydrochloride (CCH) in milk was performed utilizing the prepared Au NBs substrate, and the limit of detection was 0.01 mg/mL. In the range of 0.01~1 mg/mL, the mass concentration of CCH and SERS signal intensity satisfied the linear relationship of y=258.467x+150.501 and the value of correlation coefficient was 0.9785. In addition, the recovery of spiked samples fluctuated between 96.80% to 111.38%. These results proved that the method is simple and fast, and it is promising to be applied to the field detection of antibiotics in milk.