A mediator immobilized dimethyl sulfoxide (DMSO) sensor using DMSO reductase (DMSO-R) was constructed. Methyl viologen (MV) was used as a mediator and immobilized on a glassy carbon (GC) electrode with Nafion polymer. DMSO-R from Rhodobacter sphaeroides f. sp. denitrificans was retained by a dialysis film on the modified GC electrode. The amperometric signal in response to DMSO was observed. The linear range of the calibration curve for DMSO was between 0 and 600 µM. The response time was within 100 s and the relative standard deviation was 4% at 200 µM DMSO (n = 4). To eliminate the background noise derived from oxygen in samples, the glucose oxidase–catalase retained DMSO sensor was also examined.