Sarcosine is an important biomarker in the diagnosis of prostate cancer. It is essential to explore a method that detects sarcosine in biological samples quickly and accurately for the early screening and diagnosis of prostate cancer. Considering the high catalytic efficiency and good selectivity of enzymes, there is potential for establishing enzymatic methods for the determination of sarcosine. However, the poor stability, high economic consumption, and challenging recyclability of free enzymes limit their use. Here, sarcosine oxidase was immobilized to improve its stability. The properties of the immobilized enzyme, including the morphology and stability, were characterized. Then, the enzymatic reaction between the immobilized enzyme and the substrate sarcosine was employed to establish a fluorescence method with a linear range from 0.1563 to 10.00 μg mL⁻¹, a LOD of 0.04 μg mL⁻¹, and a recovery of 84.7–104.0%. It has been proved that sarcosine can be detected using the established method with short time consumption, high specificity, and low cost, and which is easy to operate and is affordable for many laboratories and community-level medical institutions. These results demonstrate that this method shows potential for practical applications in the diagnosis of prostate cancer.