An immobilized, cell-free system to produce isobutanol, a next-generation biofuel, was engineered to improve its production using biochemical approaches. A previously described approach involving random immobilization on suspended beads was superseded by a targeted, ordered cellulosome-based immobilization system that facilitated substrate channeling and minimized diffusion limitations. For this purpose, keto-acid decarboxylase, alcohol dehydrogenase, and formate dehydrogenase were genetically fused to a specific dockerin module, originating from different microbial species, each of which bound selectively to a complementary cohesin-bearing scaffoldin protein. Engineering of the fusions, as well as tuning the expression, was key to obtaining a working efficient multi-enzyme system. This system produced isobutanol at a titer of 5.92 g/L and 78.4% yield, which represented a marked improvement over previous results that used a cell-free method to produce this biofuel. This approach is promising, both as a starting point for further expansion of biofuel production and as a general enzyme-immobilized platform for producing biochemicals in vitro.