Background: The renal biopsy is an accurate and reliable gold standard for membranous nephropathy (MN) diagnosis. However, it is an invasive procedure involving the risk of hemorrhage or infection. Thus, an alter-native approach that can facilitate the effective diagnosis and treatment monitoring of idiopathic membranous nephropathy (IMN) is urgently needed. Methods: We established a dual-labeled time-resolved fluoroimmunoassay (TRFIA) to simultaneously detect phospholipase A2 receptor (PLA2R)-IgG4 and PLA2R-IgG antibodies. Utilizing this assay, we determined the ratio of autoantibodies in the serum of patients with different kidney diseases and normal controls. Results: The sensitivity of TRFIA for detecting anti-PLA2R-IgG and anti-PLA2R-IgG4 was 0.12 mu g/mL and 0.001 mu g/mL, respectively. Human IgA did not interfere with the assay. Compared to anti-PLA2R-IgG alone, the pos-itive rate of IMN could be increased from 86.5 % to 91.7 % through the combined use of anti-PLA2R-IgG4 and the PLA2R-IgG4/IgG ratio. In contrast, the false-positive rates for the detection of IgA nephropathy, lupus ne-phropathy, diabetic nephropathy, and minimal change nephropathy decreased from 25 to 50 % to 0 %. Conclusions: The dual-labeled PLA2R-IgG4/IgG-TRFIA for simultaneous detection of anti-PLA2R-IgG4 and anti-PLA2R-IgG will contribute to improved accuracy of IMN diagnosis.