Objective Emerging evidence suggests that the gut microbiota may play a role in sleep regulation via the microbiota–gut–brain axis; however, its relationship with objective sleep parameters and serological markers remains unclear. This study aimed to elucidate the relationship between polysomnographic, serological, and gut microbiota changes in patients with short sleep insomnia. Methods Patients with short-sleep insomnia and controls underwent polysomnography. Serum and faecal samples were collected. Serum 5-hydroxytryptamine (5-HT), brain-derived neurotrophic factor (BDNF), melatonin (MT), and orexin A (OXA) were measured by ELISA. Gut microbiota composition was analysed using 16S rRNA sequencing. Spearman’s partial correlation and redundancy analysis (RDA) were performed in the insomnia group with adjustment for relevant confounders. Results Compared with controls, insomnia patients showed significantly reduced sleep efficiency (SE), total sleep time (TST), non-rapid eye movement (NREM) and REM sleep duration, and N2 and N3 sleep, accompanied by prolonged sleep latency (SL), REM latency, and wake after sleep onset (WASO) duration and its proportion, as well as increased spontaneous microarousals during total and NREM sleep, and the frequency of sleep stage transitions and awakenings, and the relative power of theta, alpha, and beta waves during NREM sleep and delta, theta, and alpha waves during REM sleep ( p — 0.05). Serum 5-HT, BDNF, and OXA levels were elevated ( p — 0.05). While α -diversity did not differ, β -diversity differed significantly, with increased abundance of Evtepia , Anaerostipes , and Blautia_A , and reduced L-methionine salvage cycle III. Correlation analyses revealed significant associations among gut microbiota, sleep parameters, and serum markers, particularly involving N3 sleep and SL. Conclusion Short-sleep insomnia is associated with alterations in sleep parameters, serological indicators, and gut microbiota, suggesting that gut microbiota modulation may represent a potential therapeutic target.