In this study, the stereocomplexation of poly-L-lactide (PLLA) and poly-D-lactide (PDLA) was monitored in real time using fluorescence spectroscopy. To introduce fluorescence probes at the beginning (i.e., alpha position) of the PLLA and PDLA chains, a pyrene derivative was employed as an initiator for ring-opening polymerization of L,L-lactide (LLA) and D, D-lactide (DLA), respectively, and the post-polymerization functionalization of the hydroxyl end-groups of the PLLA were obtained by initiating LLA using the BuOH/Sn(Oct)(2) system to form polylactide decorated with a pyrene moiety on the opposite polymer chain-end (i.e., omega position). Using these approaches, linear Py-PLLA-OH, Py-PDLA-OH, and Bu-PLLA-Py with molar masses equal to 3100, 3350 and 3300 g mol(-1), respectively, were prepared. Next, equimolar compositions of Py-PLLA-OH/Py-PDLA-OH and Py-PDLA-OH/Bu-PLLA-Py in different solvents were prepared, and the stereocomplexation, which is related to excimer formation between the Py end-groups, was directly observed using fluorescence measurements of the excimer emission at 470 nm in both AcCN and THF solutions, respectively. The kinetic curves based on the I-E/I-M ratio as a function of time are complex in shape and indicate interaction of two enantiomeric chains occurred. In addition, at the beginning of the association, the PLA enantiomeric pairs form stereocomplex crystallites. The probability of parallel and antiparallel interactions of PLA enantiomers during stereocomplexation is similar and consistent with Cantow's calculations. In addition, the DSC analysis of the final PLA products confirmed that only stereocomplex crystallites were formed in these two solvents.