The objective of this field-scale study was to evaluate the effectiveness of controlling trichloroethylene (TCE) plume migration using the polycolloid substrate (PS) biobarrier. The developed PS (containing soybean oil, lactate and surfactants) could release substrate to enhance the TCE dechlorination. In this study, a biobarrier comprising PS injection wells was installed. Injection wells were installed at 5-m intervals, and approximately 15 L of PS was injected into each well. Results show that TCE concentrations in the injection wells dropped from an average of 87 μg/L to below 1 μg/L after 35 days of PS injection. The total organic carbon concentrations in the injection wells increased from an average of 2.1–543 mg/L after 30 days of PS injection. The dissolved oxygen (DO) concentrations and oxidation-reduction potential (ORP) values dropped from an average of 1.6 mg/L to below 0.1 mg/L and from 124 mv to −14 mv after 20 days of injection, respectively. The DO and ORP remained in anaerobic conditions during the remaining 100 days of the operational period. TCE degradation by-products were observed in groundwater samples during the operational period. This reveals that the addition of PS could effectively enhance the reductive dechlorinating of TCE.