Soil and groundwater at many existing and former industrial areas and disposal sites is contaminated by halogenated organic compounds that were released into the environment. Halogenated organic compounds are heavier than water. When they are released into the subsurface, they tend to adsorb onto the soils and cause the appearance of DNAPL (dense-non-aqueous phase liquid) pool. Among those halogenated organic compounds, trichloroethylene (TCE), a human carcinogen, is one of the commonly observed contaminants in groundwater. Thus, TCE was used as the target compound in this study. The objective of this study was to develop the emulsified carbon-releasing substrate and apply it as the filling material in the permeable reactive barrier to remediate TCE-contaminated groundwater. In this study, the developed emulsified carbon-releasing substrate contained soybean oil, lactate, biodegradable surfactant (Simple GreenTM and lecithin), and nutrients. Results of emulsion test show that up to 90% of the emulsified carbon-releasing substrate was distributed effectively in the soil pores. The emulsified carbon-releasing substrate was able to provide carbon for the enhancement of in situ anaerobic biodegradation for a long period of time. A pilot-scale study was operated at a TCE-contaminated site located in southern Taiwan. Emulsified carbon-releasing substrate emulsion was pressure-injected into the remediation wells. A total of 120 L of emulsified carbon-releasing substrate was injected into the test site. Based on the groundwater analytical results, dissolved oxygen, oxidation-reduction potential, and sulfate concentrations decreased after injection. However, the anaerobic degradation byproduct, acetic acid, increased after injection. Results also show that the total viable bacteria increased in the upgradient injection (remediation) well. Decrease in TCE concentration (dropped to below 0.01 mg/L) was also observed after substrate injection, and TCE degradation byproducts, cis-1,2-dichloroethene (cDCE) and vinyl chloride (VC) were also observed. Result of microbial analyses show that various TCE-degrading bacteria exist in the groundwater samples including Ralstonia sp., Clostridium sp., Uncultured Burkholderiales bacterium, Hydrogenophaga sp., Acidovorax sp., Zoogloea sp., Hydrocarboniphaga sp., Uncultured Curvibacter sp., Pseudomonas sp., Comamonas sp., Aquabacterium sp., and Variovorax strains. This reveals that the anaerobic dechlorination of TCE is a feasible technology at this site. Slug test result show that only a slight variation in soil permeability of the injection well was observed. This indicates that the substrate injection would not cause clogging of the soil pores. Results from the cost analysis show that the total cost for the test site remediation was approximately USD13,442 per year. This indicates that the developed system has the potential to be developed into an environmentally, economically, and naturally acceptable remedial technology. Knowledge obtained from this study will aid in designing a carbon-released substrate biobarrier system for site remediation.