The endocannabinoid system, particularly the cannabinoid receptor 1 (CB1), is essential for regulating numerous physiological processes, including pain, mood, appetite, and neurodegeneration. Given its crucial role, CB1 has become a target for therapeutic interventions with significant potential for treating various disorders. However, conventional methods such as calcium imaging and patch-clamp can only detect drug concentrations in the nanomolar to micromolar range, highlighting the need to develop more sensitive drug screening methods. To address this issue, we developed an ultra-sensitive biosensor based on cell-derived CB1 nanovesicles (CB1-NV) coupled with carbon nanotube (CNT)-printed electrodes. This ultra-sensitive sensor can detect cannabinoid compounds at picomolar concentrations by converting receptor-mediated Ca²⁺ influx into measurable electrical signals. The sensor exhibits remarkable sensitivity in terms of detecting trace THC amounts (approximately 0.001%) in hemp seed oil, which conventional methods fail to detect. Compared with conventional methods, the developed biosensor exhibited a 1,000-fold improvement in sensitivity, offering a promising tool for high-throughput drug screening and therapeutic research. The CB1-NV sensor utilizes cell-free vesicles to preserve the cellular environment; however, since live cells are not involved in the measurement process, there is no requirement to maintain cell viability.