Polymers of varying molecular weights and charges are extensively used for diverse papermaking operations such as retention, drainage, and waste treatment/recovery. Owing to the heterogeneous nature of both the polymer and the paper slurry and to the high shear environment in which paper is made, it is a challenging task to study polymer adsorption in this system and, more importantly, to be able to correlate the results with paper machine performance. In this study, the technique of fluorescence microscopy was adapted to determine the adsorption characteristics of a fluorescent cationic flocculant on three different papermaking slurries. In the absence of fillers and dissolved and colloidal substances (DCS), polymer is shown to adsorb on high surface area regions of the fiber resulting from mechanical fiber processing operations. However, in a composite alkaline furnish containing CaCO3 filler and under conditions and time scales prevalent on a papermachine, the polymer preferentially adsorbs to the filler. The filler floes in turn are adsorbed to the fibrils. In a mechanical furnish with DCS, polymer-DCS complexes are formed that alter the adsorption characteristics of the polymer. Results are discussed in terms of kinetics of adsorption, shear-induced collisions, and interactions with solution species. Furthermore, the utility of the fluorescence microscopy technique to study adsorption in complex industrial matrices is demonstrated.