High quality hydrophobic quantum dots (QDs) are increasingly popular in developing ultrasensitive immunoassays. However, the conjugation of biomolecules usually suffers from complex modifications and compromised luminescence. Herein, a novel one-step emulsification-assisted approach was described to synthesize antibody-decorated vesicles with high QDs-loading, outstanding dispersity and high quantum yields sustaining. Initially, the prerequisites for fabricating such vesicles were systematically investigated. QDs diluted in non-polar solvents were emulsified into compact nanospheres and accommodated with recognizing antibodies and stabilizing proteins that contained abundant amino groups. Then, the universality of this technique was verified by introducing three kinds of hydrophobic QDs to fabricate similar vesicles. Finally, to evaluate the applicability, an immunochromatographic assay toward 12 prohibited phosphodiesterase-5 inhibitors in health foods was developed with outstanding sensitivity (a significant analytical improvement of 5–80 folds compared to the reported ICA methods) and high accuracy. The present work highlights a novel and effective approach to produce high hydrophobic QDs-loaded immunoprobes, which is potentially generalizable to overcome limitations of weak fluorescence, low dispersity and complicated synthesis when conjugating biomolecules and hydrophobic QDs.