Melatonin secretion by the pineal gland happens predominantly at night time, though ocular publicity to brilliant mild suppresses its secretion. When measured beneath dim mild situations, the melatonin focus in plasma gives a dependable indicator of the state of a person’s central circadian pacemaker within the hypothalamus. Particularly, the onset of melatonin secretion within the night, conventionally outlined because the time when the plasma focus surpasses a threshold of 10 pg mL−1, serves as a gold-standard measure of the beginning time of physiological night time. Present melatonin detection strategies usually require costly assays and instrumentation in addition to educated personnel, or they lack ample sensitivity and anti-interference capabilities, which restrict their use in point-of-need settings. To handle these challenges, we developed a europium nanoparticle-based lateral move immunoassay (EuNP-LFIA) built-in with a 3D-printed smartphone-based fluorescence reader for on-site melatonin detection in plasma. Six key parameters, specifically readout time, EuNP dimension, antibody conjugation stage, Tween-20 focus, EuNP deposition quantity, and the focus of melatonin–bovine serum albumin on the nitrocellulose membrane, have been systematically optimized. Upon optimization, the sensing platform exhibited extremely delicate detection of melatonin in buffer answer and achieved a restrict of detection (LOD) of 9.99 pg mL−1. For melatonin-spiked plasma samples, the restoration charges ranged from 82.58% to 114.70%, indicating wonderful repeatability and reliability. General, the proposed technique possesses excessive sensitivity and accuracy, providing a transportable, speedy (<15 min), cost-effective, and user-friendly answer for real-time monitoring of plasma melatonin.