The development of molecular spectroscopy has facilitated the application of surface-enhanced Raman spectroscopy (SERS) for the detection of biomolecules, such as proteins and antibiotics. However, the sensitive and reproducible detection of SERS signals for unlabeled target antibiotic molecules remains a challenge. Here, we used bromide ions and calcium ions to improve the surface state of silver (Ag) nanoparticles, eliminate the interference of citrate ion signals, and perform an indiscriminate analysis of the different types of antibiotics. Calcium ions also act as aggregating agents to induce the "hot spots" formed by Ag nanoparticles, which can accommodate the entry of antibiotic molecules to realize high-sensitivity detection of antibiotic molecules. Using this method, we obtained the SERS spectra of antibiotic molecules with good reproducibility and a high signal-to-noise ratio. Also, using dichloromethane as the internal standard, we proposed, for the first time, the use of changes in peak intensity to distinguish between different antibiotic molecules and characterize changes in the structure and properties of these antibiotic molecules. Additionally, we detected the Raman signal of antibiotic molecules at low concentrations in human serum successfully. This method will find wide applications in the fields of medical therapy and biological macromolecular analysis.