Insomnia is a common sleep disorder with a high prevalence and substantial adverse consequences. There is growing interest in identifying novel therapeutics from herbal medicine. Tenuifolin is a major constituent of the well-known anti-insomnia herb Radix Polygala. The present study investigated the neural activity in response to tenuifolin during rest/wake behaviour in zebrafish and identified the potential biological signalling pathways involved. An automatic video tracking system was used to monitor the behavioural response of zebrafish larvae for 24 h after treatment with tenuifolin. In total, six rest/wake parameters were measured and visualized with a behavioural fingerprint. Time series analysis was conducted by averaging the total rest and waking activity in 10 min intervals. A correlation analysis was performed between tenuifolin and well-known compounds to analyse the underlying biological signalling pathways. Reverse transcription-quantitative PCR was also performed to detect the effects of tenuifolin on the transcription of interesting genes associated with the signalling pathways that were potentially involved. The present results suggested tenuifolin significantly increased the total rest time during the dark phase, with a slight effect on the waking activity in zebrafish larvae. This behavioural phenotype induced by tenuifolin is similar to that of selective serotonin reuptake inhibitors and gamma-aminobutyric acid (GABA) agonists. Furthermore, the expression levels of GABA transporter 1 were significantly increased after tenuifolin treatment. No significant difference was determined in other associated genes in untreated control and tenuifolin-treated larvae. The present results suggested that tenuifolin caused sleep-promoting activity in zebrafish and that these effects may be mediated by the serotoninergic systems and the GABAergic systems.