The labelling reagent CdSe@CdS-QDs-Cys (QDs-Cys) with the grain diameter of 4.5 nm was synthesized by modifying CdSe@CdS quantum dots (QDs) with cysteine (Cys). At the same time, QDs-Cys-Ab(IgE), a phosphorescent quantum dot probe, was developed based on the labelling reaction between -COOH of QDs-Cys and -NH(2) of goat anti human IgE antibody (Ab(IgE)). This probe with excellent biocompatibility and high specificity could not only emit strong and stable room temperature phosphorescence (RTP), but also could carry out specific immunoassay (IA) with immunoglobulin E (IgE), causing the RTP of the system to sharply enhance. Thus, a new solid substrate room temperature phosphorescence immunoassay (SSRTPIA) for the determination of IgE was established. The limit of quantification (LOQ) of the method was 0.12 fg spot(-1), corresponding concentration was 3.0 × 10(-13) g mL(-1) and sampling quantity was 0.40 μL spot(-1). This highly selective, sensitive and accurate SSRTPIA has been applied to determine IgE in biological samples and diagnose diseases, and the results agreed well with those obtained by enzyme-link immunoassay (ELISA). Meanwhile, the mechanisms of QDs-Cys labelling Ab(IgE) and the determination of IgE by SSRTPIA were also discussed.