Bioavailability is a key parameter in assessing contaminant transfer to biota. However, the input patterns and soil use types may impact the metal bioavailability. Several soil parameters were measured including chemical properties, such as pH, organic C, and Cu solution/solid speciation, and biological properties, such as soil microbial biomass C (SMBC), seed germination, and root elongation, to evaluate the bioavailability of Cu contaminated soils from three different sources, i.e., non-ferrous metal mining, Cu-based fungicides, and Cu-smelting. The results revealed that free Cu2+ ion in soil solution and the ratios of Cu fractions to total Cu content in the solid phase could not be used to predict total Cu content in soils. The indexes of seed germination and root elongation appeared not to be good biomonitors of Cu contamination in soils, which were more sensitive to soil pH and soil organic carbon (SOC). Relationships between SMBC and soil Cu forms or the ratio of SMBC/SOC and soil Cu forms showed that free Cu2+ ion and humic acid-complexed Cu could significantly inhibit soil microbial activities. Our findings suggested that both metal chemical forms and biological bioassays should be considered as a complementary technique rather than an alternative to evaluate the metal bioavailability from different pollution sources.