Immune checkpoint blockade therapies are promising next generation immunotherapeutic treatments for cancer. Whilst sequential solid biopsies are an invaluable source of prognostic information, they are not feasible for monitoring therapeutic outcomes over time. Monitoring soluble immune checkpoint markers expression in body fluids could potentially be a better alternative. Current methods (e.g. ELISA) for detecting immune-checkpoint proteins mostly rely on the use of monoclonal antibodies which are expensive and time-consuming to manufacture and isolate. Herein, we report an integrated surface enhanced Raman scattering (SERS)-microfluidics device for the detection of immune checkpoint proteins which involves the use of i) nano yeast single chain variable fragment (scFv) as a promising alternative to monoclonal antibodies providing high stability at relative low-cost and simplicity for production, ii) graphene oxide functionalised surface to reduces the bio functionalization steps, thus avoiding the general paradigm of biotin-streptavidin chemistry and iii) a microfluidic platform enabling alternating current electrohydrodynamics (ac-EHD) induced nanomixing to enhance the target scFv binding and minimize the non-specific interactions. Specific and multiplex detection of immune checkpoint biomarkers is achieved by SERS based spectral encoding. Using this platform, we successfully demonstrated the detection of clinically relevant soluble immune checkpoints PD-1, PD-L1 and LAG-3 from as low as 100 fg/mL of analytes spiked in human serum. •A single chip for simultaneous detection of multiple immune checkpoint biomarkers.•Nanoyeast-scFv probes as the alternative to monoclonal antibodies.•Graphene oxide nanosheets as efficient functionalisation material.•Ac-EHD and SERS encoding provide highly specific and multiplex detection.•Highly sensitive detection of target antigen in human serum (100 fg/mL).