In this study, a compact disc (CD) microfluidic device based on reciprocating flow is developed for the rapid discrimination of single-nucleotide mismatch (SNM). This device consists of a PDMS CD slab containing eight SNM assay units and a glass substrate immobilized with DNA probe array. The rapid SNM assay is realized by two-step controllable reciprocating flow in the microchannel, resulting from centrifugal force and capillary action as well. A perfect-match probe and three mismatched probes containing a SNM at various positions were designed to investigate the ability of this device to discriminate SNM. Based on the difference between the dissociation kinetics of perfect-match and mismatched duplexes, this CD device was able to discriminate SNM within 400 s. Moreover, only sub-microliter volume DNA samples (350 nL) and washing buffers (350 nL) were required per assay unit. The results indicate that this device has the potential to be used for fast, accurate gene diagnostics, especially for the detection of SNPs.