Fluorescence correlation spectroscopy based on upconverting nano-particles (UCNPs) can detect single particles in strongly autofluorescent samples, such as plasma. Nanoparticle aggregation, however, is a problem in correlation spectroscopy measurements, and biological media are known to induce aggregation. Here we present an improved UCNP surface chemistry and use these UCNPs in an upconversion cross-correlation spectroscopy (UCCS)-based homogeneous immunoassay for thyroid-stimulating hormone, where the simultaneous emission of green- (NaYF4 :Yb3+,Er3+ ) and blue- (NaYF4 :Yb3+,Tm3+) emitting UCNPs is detected when they are bound together by the analyte. The improved coating suppresses UCNP aggregation, even in plasma samples. Additionally, increasing the Tm doping of blue-emitting UCNPs enhances their brightness at a high excitation intensity and also results in a significantly shorter luminescence decay time, which improves the probability of detecting coincident blue and green emission from the bound UCNPs in the UCCS immunoassay.