Recently, nonmetallic substrates have stimulated great interest in surface-enhanced Raman scattering (SERS)-based immunoassay owing to their good uniformity, stability, and biocompatibility. In this context, a novel nonmetallic SERS-based immunoassay mediated by two-dimensional molybdenum disulfide (MoS2) was delivered for the sensitive and specific monitoring of carbohydrate antigen 19–9 (CA19-9). The effective enrichment of molecules on the large active surfaces of MoS2 as well as potential 532-nm laser-induced charge transfer resonances between them engendered desirable enhancement factor values at the level of 105. Intriguingly, a sandwich immunocomplex combined MoS2 nanoflower and nanosheet exhibited not only a wide linear range from 5 × 10−4 to 1 × 102 IU·mL−1 but also a limit of detection as low as 3.43 × 10−4 IU·mL−1 towards CA19-9. More meaningful, the analytical result for clinical patient serum sample was basically compared with the conventional chemiluminescent immunoassay. Such a novel nonmetallic SERS-based immunosensor with desirable biocompatibility and sensitivity is promising for clinical diagnosis.