The low efficiency of enzymes used in the bioprocessing of biomass for biofuels is one of the primary bottlenecks that must be overcome to make lignocellulosic biofuels cost-competitive. One of the rate-limiting factors is the accessibility of the cellulase enzymes to insoluble cellulolytic substrates， facilitated by surface absorption of the carbohydrate-binding modules (CBMs)， a component of most cellulase systems. Despite their importance， reports of direct observation of CBM function and activity using microscopic methods are still uncommon. Here， we examine the site-specific binding of individual CBMs to crystalline cellulose in an aqueous environment， using the single molecule fluorescence method known as Defocused Orientation and Position Imaging (DOPI). Systematic orientations were observed that are consistent with the CBMs binding to the two opposite hydrophobic faces of the cellulose microfibril， with a well-defined orientation relative to the fiber axis. The approach provides in situ physical evidence indicating the CBMs bind with a well-defined orientation on those planes， thus supporting a binding mechanism driven by chemical and structural recognition of the cellulose surface.