Highly selective and sensitive determination of dopamine (DA) from multiple sources remains a persistent and significant challenge. Here, we develop an imidazole-triggered in situ fluorescence reaction system for highly selective and sensitive determination of DA from various sources (human, horse, dog, rabbit, and mouse). The system operates by catalyzing the oxidation of DA with 1,5-Dihydroxynaphthalene (1,5-DHA) through a Lewis base formed by imidazole, leading to the rapid generation of yellow azamonardine fluorescent compounds (AFC). Notably, the system demonstrates minimal background noise and a high signal-to-noise ratio of up to 300-fold with a determination limit of 33.33 pM, making it 10-100 times more sensitive than conventional enzyme-linked immunosorbent assay (ELISA) methods. Moreover, selectivity tests reveal that our system can effectively distinguish between several common interfering substances, even at concentrations as low as 10 nM. The developed system shows promising results in detecting DA from diverse sources (humans, horses, dogs, rabbits, and mice), including urine samples from clinical patients, exhibiting good agreement with traditional ELISA kits. Therefore, the established in situ fluorescence reaction system holds great potential for the determination of DA-related disorders due to its impressive analytical capabilities.