Biosynthesis of n-caproate through chain elongation has attracted increasing attention in the past years. However, the low titers have limited its wide application. To improve the production of n-caproate, the influence of process parameters on caproate fermentation was comprehensively investigated in the cell-immobilized system. Batch tests showed that the caproate production was remarkably affected by the ethanol-to-acetate ratio, the concentration of substrates, and the pH. The corresponding optimum values of these three parameters were 8:3, 660 mmol/L, and 7.0–7.4, respectively. The immobilized cells preferred to use the mixture of acetate and butyrate as electron acceptors, obtaining the highest caproate production of 146.6 mmol/L (17.0 g/L) with the acetate/butyrate ratio of 1:1. Analysis of biomass weight, thermogravimetry/differential scanning calorimetry, and Fourier-transform infrared spectroscopy illustrated that the immobilized cells dominated the total amount of cells in the immobilization system. The extracellular polymeric substance played an important role in improving the activity of the biofilm. The feasibility of high caproate production from real wastewater was finally validated. This study provides an insight into the development of an efficient and cost-effective way for caproate production from waste biomass.