Ideal urine screening should realize the detection of a variety of different urine proteins, corresponding to the diagnosis of different diseases. However, there is a lack of effective instruments to realize the simultaneous screening of albumin and light chain protein. In this paper, a specialized multiplexed urine protein detection instrument based on SPR technology was developed for quantitative detection of human serum albumin (HSA), beta-2-microglobulin (B2M), kappa light chain and lambda light chain protein. A modified BSA sensor membrane was developed to realize one-step direct detection of the analyte. The sensitivity, repeatability and specificity of the SPR instrument were analyzed and tested, and the standard curves of four proteins were calculated. Comparing the results between healthy people and nephrotic patients by our instrument, it was found that there was a significant difference in the two groups for HSA and B2M detection (p < 0.01), indicating that HSA and B2M are suitable markers for nephropathy diagnosis. Furthermore, the performance of our instrument was compared with commercialized platforms and got lower experimental concentrations than the immunoturbidimetry method in kappa and lambda detection. It will promote early screening of kidney and tumour diseases simultaneously by applying our SPR sensor in the future.