The unique optoelectronic properties of I-III-VI2 nanocrystals (NCs) have attracted extensive attention. Herein, element Se in oleylamine reduced by alkythiol, which has been demonstrated to generate highly reactive alkylammonium selenide, was selected as the Se precursor by us to successfully synthesize high-quality tetragonal AgGaSe2 NCs via a facile colloidal method in just 2 minutes. Further, the photoluminescence (PL) properties of the as-synthesized AgGaSe2 NCs were systematically optimized through utilizing one Zn precursor to integrate shell coating and anionic/cationic alloying strategies into our reactive system, resulting in not only the obvious improvement of PL intensity but also tunable PL color from blue to red. Furthermore, the ligand exchange approach was adopted for the aqueous phase transfer of the oleophilic AgGaSe2/ZnSe NCs. Our data suggest that either metalated mercaptopropionic acid (Zn-MPA) short- or 11-mercaptoundecanoic acid long-chain ligand exchanged NCs all could maintain the original high crystallinity, present good water solubility, and retain up to nearly 95% and 70% of the initial PL intensity, respectively. Benefiting from the low cytotoxicity, the water-soluble AgGaSe2/ZnSe NCs can be applied as a fluorescent probe in cell imaging and signal labels for the fluoroimmunoassay of prostate-specific antigen, implying their potential in biological application.