Early detection of cancer biomarkers provides clinically valuable information. While the conventional enzyme-linked immunosorbent assay (ELISA) has been routinely used for individual cancer markers, methods for simultaneous determination of multiple markers within a single sample are still in demand. Here, we present a novel oligonucleotide-linked immunosorbent assay (OLISA) with a multiplexing capability on the same microwell plate-based system as in ELISA. Employing a DNA oligonucleotide that is covalently conjugated to the detection antibody and a complementary RNA oligonucleotide which is appended with a fluorophore and a quencher, degradation of the RNA in the DNA-RNA duplex by RNase H is exploited for fluorescent signal generation. Iterative cycles of DNA-RNA duplexation and subsequent degradation of the RNA in the duplex by RNase H further lead to amplification of the detection signal in OLISA. Moreover, the use of antibody-oligonucleotide conjugates enables multiplexing of OLISA, which is successfully demonstrated by tethering DNA molecules to detection antibodies and by performing assays for three common cancer markers including α-fetoprotein, prostate-specific antigen, and carcinoembryonic antigen. With the simple procedure and reliable detection performance, the developed multiplex OLISA has a wide potential for use in analysis of a panel of biomarkers in clinical diagnostics.