Cancer biomarkers have been facing some issues such as poor accuracy and low sensitivity in the early diagnosis of tumors. Utilizing biotin-labelled peptide as a mass tag (MT), this work proposes a high-throughput biosensing strategy for matrix-assisted laser desorption/ionization-time of flight mass spectrometric (MALDI-TOF-MS) immunoassay of multiple lung cancer biomarkers. Due to little required dosage, satisfied stability, high sensitivity and accuracy, this method can achieve off-site centralized signal detection after on-site sample incubation. The proposed approach has been successfully applied for the detection of carcinoembryonic antigen (CEA), carbohydrate antigen199 (CA199), carbohydrate antigen 125 (CA125) and cytokeratin-19-fragment (CY211) in serum samples from various stages of non-small cell lung cancer. Based on the analysis of multiple parameters and pathological results, significant differences in biomarkers are found in serum samples of lung cancer patients at different stages. More importantly, the analysis of multiple tumor biomarkers can improve the accuracy and sensitivity of early diagnosis. Therefore, the multiple immunoassay based on MALDI-TOF MS exhibits exceptional performance in terms of high throughput, little sample dosage, stability and sensitivity.