The efficiency of many anesthetic regimens is controversial, with side effects especially in the vulnerable children and old population. The study aimed to evaluate the safety and efficacy of low-dose combination of ketamine, fentanyl, and dexmedetomidine (KFD) for anesthesia and analgesia in the neonatal and elderly rats. KFD rapidly induced anesthesia and analgesia in either postnatal days 6 (P6) or 13 months (13M) old rats. Meanwhile, KFD administration had no adverse effects on the cardiovascular and respiratory systems. Compared with control group, there were no distinct morphologic changes in kidney, liver, and brain in KFD group. Moreover, administration of KFD had no influence on hepatic and renal function in rats of both ages. Furthermore, there was no obvious difference in cognitive function between control and KFD groups. These results indicated that the administration of KFD combination offered safe and efficient anesthesia. Collectively, our results suggest the potential implication of the KFD combination in anesthesia management.