The thin film geometry of block copolymers (BCPs) required by many applications often leads to a shift of their order-to-disorder transition temperature (T_ODT) due to the presence of air–polymer and substrate–polymer interfaces. A challenge associated with measuring BCP thin film T_ODTs is the lack of a simple, generalizable, non-destructive characterization method, which is of critical importance for exploiting BCP self-assembly in nanotechnology. In this work, we present a non-invasive fluorescence characterization method for determining the T_ODT of BCP thin films. Fluorescent pyrene molecules were covalently attached at nearly trace levels to one block and used as a probe of the local polymer matrix. The BCP T_ODT can be determined by a discontinuous change in the intensity ratio I₁/I₃ of pyrenyl vibronic emission bands as a function of temperature, which is induced by a change of the nanoscopic chemical environment around the pyrene labels at the ODT. When the BCP film thickness is sufficiently small, an increase of T_ODT compared to bulk is observed. This increase of T_ODT in thin films is mainly attributed to attractive polymer–substrate interactions, the effects of which can be varied by adjusting the film thickness and substrate surface hydrophobicity. These results not only demonstrate that fluorescence characterization can be used as a generalizable approach for determining BCP thin film ODTs, but also provides additional insights on manipulating functional BCP nanopatterns by controlling the substrate surface functionality.