Though traditional fluorescence analytical methods have excellent luminescence properties including good selectivity and sensitivity, steady fluorescence and convenient preparation, they still have some insurmountable disfigurements, such as photo bleaching of majority fluorescence reagent, unsteadiness of fluorescence signal, the side effect of fluorescence reagent to body cells. So we prepared steady composite nanoparticles as the fluorescence probes for the determination of trace substance with the advantages of easy preparation and good dispersity.In this paper, we successfully prepared composite fluorescence nanoparticles and use them as fluorescence probes to determine proteins and pH. The details are summarized as follows:(1) Water-in-oil microemulsion is a widely used method of preparing nanomaterials of small size. With this reversed micelles method, we prepared nano-pH sensor based on fluorescent silica nanoparticles developed by encapsulating dye at room temperature. We also prepared a fluorescence ratiometric nano-pH sensor based on dual-fluorophore-doped silica nanoparticles. These newly prepared nanoparticles have been tested for pH determination in real water samples with good results. The methods in this paper proved to be simple, rapid, and specific, and the recovery and relative standard deriation are very satisfactory.(2) Under ultrasonic irradiation, we successfully synthesized Tb-Eu/acac / PAM composite nanoparticles. The synthesis of these nanoparticles led to a particularly simple, inexpensive and sensitive method. These nanoparticles are highly resistant to photobleaching and emit narrow and steady fluorescence and employed as a fluorescence probe for the determination of proteins.