Living cells are constantly threatened by endogenous and environmental agents that can induce various DNA lesions including 8-oxoguanine (8-oxoG). Increasing evidence has suggested that 8-oxoG is not only a biomarker of oxidative stress, but also a novel epigenetic-like modification involved in transcriptional regulation in mammalian cells. Measurement of DNA damage and repair is useful for both basic research and clinical applications, but current methods for 8-oxoG detection still suffer from some problems such as poor selectivity, time consuming and being expensive. Here, we developed a fast and simple biosensing approach for quantitative analysis of 8-oxoG in DNA, which was based on the selective chemical biotinylation of 8-oxoG in conjunction with biotin-streptavidin enzyme-linked immunosorbent assay. We have also successfully applied this method to achieve efficient detection of the repair activities of DNA glycosylases Fpg and hOGG1 toward 8-oxoG in vitro and in human cells. This newly developed biosensing assay should be generally applicable for rapid detection of 8-oxoG and its repair in other organisms.