As an important vertebrate model organism, zebrafish are typically studied at the embryonic stage to take advantage of their properties of transparency and rapid development. However, more and more studies require assays to be done on adults. Consequently, a good anesthetic is needed to sedate and immobilize the adult zebrafish during experimental manipulation. To date, MS-222 (tricaine methanesulfonate) is the only Food and Drug Administration approved anesthetic for aquaculture and is widely used by the zebrafish research community. Nevertheless, in adult zebrafish, MS-222 reduces heart rate and causes high mortality under long-term sedation. Consequently, adult zebrafish have limited research applications. In this study, we present a new anesthetic formula for the adult zebrafish that results in minimal side effects on its physiology under prolonged sedation. The combined use of MS-222 with isoflurane effectively extended the time of anesthesia, and the zebrafish recovered faster than when anesthetized with the traditional MS-222. Moreover, MS-222 + isoflurane did not cause reduction of heart rates, which enabled long-term electrocardiogram recording and microscopic observation on the adult zebrafish. Taken together, the new MS-222 + isoflurane formula will facilitate general applications of adult zebrafish in time-consuming experiments with minimal side effects on the model organism's overall physiology.