Carcinoembryonic antigen (CEA) is a broad-spectrum tumor marker used in clinical applications. The primarily clinical method for measuring CEA is based on chemiluminescence in serum during enzyme-linked immunosorbent assays (ELISA) in 96-well plates. However, this multi-step process requires large and expensive instruments, and takes a long time. In this study, a high-throughput centrifugal microfluidic device was developed for detecting CEA in serum without the need for cumbersome washing steps normally used in immunoreactions. This centrifugal microdevice contains 14 identical pencil-like units, and the CEA molecules are separated from the bulk serum for subsequent immunofluorescence detection using density gradient centrifugation in each unit simultaneously. To determine the optimal conditions for CEA detection in serum, the effects of the density of the medium, rotation speed, and spin duration were investigated. The measured values from 34 clinical serum samples using this high-throughput centrifugal microfluidic device showed good agreement with the known values (average relative error = 9.22%). These results indicate that the high-throughput centrifugal microfluidic device could provide an alternative approach for replacing the classical method for CEA detection in clinical serum samples.