Due to the controllable length of the fiber and its unique optical performance, the detection of the fiber SERS substrate is flexible and simple. In this study, silver nanoparticles were modified on the fiber end face using an oil-water separation experimental method, proving that this method can prepare the fiber SERS substrate and effectively enhance Raman signals. We used Crystal violet solution as the analyte to characterize the SERS fiber matrix, and studied the uniformity, sensitivity and stability of the fiber SERS substrate. In practical application, optical fiber substrate was used to detect 4-aminonenenebb thiophenol, which is widely used in the synthesis of Rhodamine 6G and pesticide intermediates. These experimental results demonstrate that the self-assembly method provides a feasible approach for the preparation of fiber SERS substrates.