Detecting biomarkers is an efficient method to diagnose and monitor patients’ stages. For more accurate diagnoses, continuously detecting and monitoring multiple biomarkers are needed. To achieve point-of-care testing (POCT) of multiple biomarkers, a smartphone biosensor system with the multi-testing-unit (SBSM) based on localized surface plasmon resonance (LSPR) integrated multi-channel microfluidics was presented. The SBSM could simultaneously record nine sensor units to achieve the detection of multiple biomarkers. Additional 72 sensor units were fabricated for further verification. Well-designed modularized attachments consist of a light source, lenses, a grating, a case, and a smartphone shell. The attachments can be well assembled and attached to a smartphone. The sensitivity of the SBSM was 161.0 nm/RIU, and the limit of detection (LoD) reached 4.2 U/mL for CA125 and 0.87 U/mL for CA15-3 through several clinical serum specimens testing on the SBSM. The testing results indicated that the SBSM was a useful tool for detecting multi-biomarkers. Comparing with the enzyme-linked immunosorbent assays (ELISA) results, the results from the SBSM were correlated and reliable. Meanwhile, the SBSM was convenient to operate without much professional skill. Therefore, the SBSM could become useful equipment for point-of-care testing due to its small size, multi-testing unit, usability, and customizable design.