Objective To screen for biomarkers in urine from patients with steroid-resistant nephrotic syndrome (SRNS) using surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) Proteinchip technology. Methods Urine samples from 9 SRNS patients, 32 steroid-sensitive nephrotie syndrome (SSNS) patients and 45 normal controls were analyzed using UA gold chip. Proteomic spectra were generated by mass spectrometry. Results Four differentially expressed biomarkers were identified with relative molecular weight of 6 703, 7 212, 11 820, 14 356. It was found that these protein peaks with relative molecular weigh of 7 212, 11 820, 14 356 were highly expressed in SRNS and 6 703 were lowly expressed in SRNS. The diagnostic cast that is constructed with these four protein to differentiate SRNS from SSNS with sensitivity of 88.89% and specificity of 93.75%. Conclusions SELDI-TOF-MS Proteinchip technology is a non-invasive, quick, easy, and convenient, and high-throughput analyzing method capable of screening several biomarkers from the urines of SRNS patients and has better clinical value.