Research on the intracerebral mechanism of insomnia induced by serotonin (5-HT) deficiency is indispensable. In order to explore the effect of 5-HT deficiency-induced insomnia on brain regions related to memory in rats, we designed and fabricated a microelectrode array that simultaneously detects the electrical activity of the dorsal raphe nucleus (DRN) and hippocampus in normal, insomnia and recovery rats in vivo. In the DRN and hippocampus of insomnia rats, our results showed that the spike amplitudes decreased by 40.16 and 57.92%, the spike repolarization slope decreased by 44.64 and 48.59%, and the spiking rate increased by 66.81 and 63.40%. On a mesoscopic scale, the increased firing rates of individual neurons led to an increased δ wave power. In the DRN and hippocampus of insomnia rats, the δ wave power increased by 57.57 and 67.75%. Furthermore, two segments’ δ wave slopes were also increased in two brain regions of the insomnia rats. Our findings suggest that 5-HT deficiency causes the hyperactivity of neurons in the hippocampus and DRN; the DRN’s firing rate and the hippocampal neuronal amplitude reflect insomnia in rats more effectively. Further studies on alleviating neurons affected by 5-HT deficiency and on achieving a highly effective treatment for insomnia by the microelectrode array are needed.