Amitriptyline is a commonly used tricyclic antidepressant (TCA) inhibiting serotonin and norepinephrine reuptake. The exact CNS action of TCAs remains poorly understood, necessitating new screening approaches and novel model organisms. Zebrafish (Danio rerio) are rapidly emerging as a promising tool for pharmacological research of antidepressants, including amitriptyline. Here, we examine the effects of chronic 2-week exposure to 10 and 50 μg/L amitriptyline on zebrafish behavior and monoamine neurotransmitters. Overall, the drug at 50 μg/L evoked pronounced anxiolytic-like effects in the novel tank test (assessed by more time in top, fewer transition and shorter latency to enter the top). Like other TCAs, amitriptyline reduced serotonin turnover, but also significantly elevated whole-brain norepinephrine and dopamine levels. The latter effect was not reported in this model previously, and accompanied higher brain expression of tyrosine hydroxylase (a rate-limiting enzyme of catecholamine biosynthesis), but unaltered expression of dopamine-β-hydroxylase and monoamine oxidase (the enzymes of dopamine metabolism). This response may underlie chronic amitriptyline action on dopamine and norepinephrine neurotransmission, and contribute to the complex CNS profile of this drug observed both clinically and in animal models. Collectively, these findings also confirm the important role of monoamine modulation in the regulation of anxiety-related behavior in zebrafish, and support the utility of this organism as a promising in-vivo model for CNS drug screening.