Cancer became a global public health problem and one of the most causes of death, and early diagnosis will decrease mortality and extend lifespan of patients. In this study, the simultaneous detection of four tumor markers in lung cancer (alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), cytokeratin-19-fragment (Cyfra21-1)) was achieved for the first time using immune sandwich structures coupled with generation collection (GC) mode of scanning electrochemical microscopy (SECM). The proposed method exhibited excellent performance in quantitative detection of the four target proteins. A good linear correlation between the signal current issued from reduction of p-benzoquinone (BQ) oxidized from hydroquinone (H(2)Q) and the amount of target tumor markers at logarithmic protein concentrations ranging from 5 ng/mL to 1 mu g/mL was achieved. The detection limit was also low, meeting the needs of clinical use. The specificity was satisfactory and signal current of the target protein was unaffected by other simultaneously detected target proteins and common interfering species. Overall, the proposed method looks promising for high-throughput protein determination based on SECM, which could potentially be applied in clinical lung cancer diagnosis.