Initial-stage prenucleation clusters (PNCs) are critical in calcium phosphate (CaP) biomineralization and thus the formation mechanisms of human bones and teeth. However, several features of PNCs require further examination, e.g., structure, ionic stoichiometry, kinetics, thermodynamics, and nucleation mechanism. In this study, we used poly(acrylic acid) (PAA)-Ca(Eu) complexes with partial Eu3+ substitution as pre-PNCs and established a fluorescence method to study PNC formation in situ based on Eu-O charge-transfer transitions. The kinetics and thermodynamics of PNC formation were explored by probing the fluorescence changes of Eu-O charge-transfer transitions during bonding between the pre-PNCs and PO43-. PNC formation was consistent with the pseudo-second-order kinetic and Langmuir isothermal adsorption models. The flexible structures of PNCs aided in regulating the subsequent nucleation and crystallization. This study provides an in situ fluorescence probing method with critical guiding significance in addressing the features of PNC formation, in addition to biomineralization.