CuNi/Fe LDHs with HRP-like activity (nanozyme) have been prepared. Contrary to the expected design, free dopamine (DA) was found to greatly enhance the catalytic performances of CuNi/Fe LDHs nanozyme. As far as we know, this is the first report that free DA boosts the catalytic performances of LDHs. Given the superior HRP-like enzyme activity of DA-CuNi/Fe LDHs, a colorimetric method for DA and tyrosinase (TYR) assay with high sensitivity and specificity was established, and it was successfully applied to quantify DA in artificial cerebrospinal fluid and TYR in newborn calf serum. The acquired insights in DA-CuNi/Fe LDHs will contribute to future rational design of other high-performance nanozymes. In addition, the novel DA and TYR assay pave a way for designing further nanozymes-based colorimetric chemo/biosensors.