Rapid, simple, and reliable detection of toxics at trace level is of great significance for public health. Surface‐enhanced Raman spectroscopy (SERS) as a powerful vibrational spectroscopic technique for molecules probing provides great advantages in trace detection; however, the low reproducibility of SERS signals from spot to spot and substrate to substrate remains a great challenge restricting its real applications. Here, we report a facile method for reproducibly preparing highly uniform self‐assembled monolayers of gold nanostars (Au NSs SAM) as SERS substrates using the classic Langmuir–Blodgett technique and introduced a statistical method to explore the signal reproducibility by t test. Our results show that the Au NSs SAM substrates fabricated in the same batch and different batches generated constant SERS signals without statistical differences and could detect dye molecules CV, R6G, and RhB in solution at concentrations as low as 10−8, 10−8, and 10−7 M, respectively. We then applied the Au NSs SAM for trace detection of Tetracycline (TC), one of the most widespread antibiotics entering the food chain and threatening the ecological balance. A detection limit of 0.05 μg/ml was achieved, with a linear response between SERS signal and TC concentration in the range of 0.05–10 μg/ml. Overall, this uniform Au NSs SAM could be reproducibly fabricated to provide a reliable platform for trace detection of TC residues or other toxics in water and is expected to have broad applications in different fields including environmental monitoring, food security, and so on.