Depression, a prevalent psychiatric disorder that significantly impacts quality of life. Cholinergic transmission dysregulation potentially contributes to emotional and behavioral disturbances. While acetylcholinesterase is commonly studied in depression, the role of butyrylcholinesterase (BChE) remains unclear. We recently developed the butyrylcholinesterase (BChE)-activatable molecular sensor (BCC) for real-time visualization of BChE activity, demonstrating its efficiency in vitro and in tumor-bearing mice. In this translational study, we employed BCC to investigate BChE changes in depression. First, we assessed BChE level in rats exposed to chronic unpredictable mild stress (CUMS), and second, in patients with depression (32 unipolar depression, 21 bipolar disorder with depressive episodes) compared to controls. Additionally, changes with respect to treatment were reassessed (n=15). Results indicated that both rats exposed to CUMS and individuals with depression exhibited significantly lower total luminescence, BChE level, compared to their controls (p < 0.001, p < 0.001). Furthermore, rats treated with fluoxetine during CUMS showed an increasing trend, although not reaching significance (p = 0.54). The significant increase in BChE level compared to baseline was observed in patients with depression at the 8th week of treatment (n=24, p<0.001). This increase was also significantly predicted by the Hamilton Depression Scale score (n=15, p=0.02). This translational study showed that there was a decrease in BChE levels in both the clinical population and the depression animal model, and that BChE levels reversed with the alleviation of symptoms. Our findings propose BChE can be a promising biomarker associated with depression diagnosis and severity.