To screen suitable substrate of Constructed Wetlands (CWs) for Cr(VI) removal, six different kinds of substrate (paddy soil, peat, red loam, sand, diatomite and fly ash) were tested using a microcosm experiment. The Cr removal efficiency, together with pH, Eh, organic content and plant biomass, was analyzed. The results showed that Cr removal efficiency in the CWs using peat and paddy soil as substrate were significantly higher than those in other CWs. The Cr(VI) concentration reached 0.003 mg/L and 0.023 mg/L in the CWs with peat and paddy soil substrates when influent Cr(VI) was 5 mg/L. The six kinds of substrate had different pH values. In general, diatomite and fly ash with high pH values were unfavorable for Cr(VI) removal. The CWs with peat and paddy soil substrates had higher organic content than the CWs with other substrates, thereby enhancing the Cr reduction and removal. Moreover, the plant biomass was significantly higher in the CWs with peat and paddy soil substrates than those in the CWs with other substrates (p < 0.05). This finding indicated that peat and paddy soil were favorable for plant growth and enhanced plant action for Cr(VI) purification. Therefore, peat and paddy soil are proposed to be used as substrates in CWs for Cr(VI) removal.