Melatonin secretion by the pineal gland happens predominantly at night time, though ocular publicity to shiny gentle suppresses its secretion. When measured below dim gentle circumstances, the melatonin focus in plasma supplies 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 skilled personnel, or they lack enough sensitivity and anti-interference capabilities, which restrict their use in point-of-need settings. To deal with these challenges, we developed a europium nanoparticle-based lateral circulate 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, had been systematically optimized. Upon optimization, the sensing platform exhibited extremely delicate detection of melatonin in buffer resolution 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 methodology possesses excessive sensitivity and accuracy, providing a transportable, speedy (