Non-toxic, highly sensitive InP quantum dot (QD) fluorescent immunoassay probes are the promising biomedical detection modalities due to their unique properties. However, InP-based QDs are prone to surface oxidation, and the stability of InP QD-based probes in biocompatible environments remains a crucial challenge because of the contradiction between lattice stress relaxation and thick shell growth. Herein, we developed thick-shell InP-based core/shell QDs by inserting a ZnSeS alloy layer, which effectively facilitated lattice stress relaxation and passivate defect states. The synthesized InP/ZnSe/ZnSeS/ZnS core/alloy shell/shell QDs (CAS-InP QDs) with nanostructure tailoring revealed a larger size, high PLQY and high optical stability. After amphiphilic polymer encapsulation, the aqueous CAS-InP QDs presented an almost constant fluorescence attenuation and stable PL intensity under different temperature, UV radiation and pH solutions. To further improve the sensitivity, the biotin-streptavidin (Bio-SA) system was firstly introduced in the fluorescence-linked immunosorbent assay (FLISA). Consequently, the CAS-InP QDs-based SA-Bio sandwich FLISA realized the detection of C-reactive protein (CRP) with the impressive limit of detection (LOD) of 0.83 ng mL−1. It is believed that stable and sensitive InP QD fluorescent probes will drive the rapid development for future eco-friendly, cost-effective and sensitive in vitro diagnostic kits.