Small molecules have been widely developed as drugs, dyes, and research tools. DNA logic system interaction with small molecules as a new analytical method has offered remarkable promise for disease diagnosis and therapy. However, tedious modification process and large sample consumption of traditional DNA logic gates limit the application of this method in bioanalysis. Therefore, developing a simple and less sample consumption platform for DNA logic gates construction is of great importance. Here, we proposed a DNA colorimetric multilevel circuit construction strategy using gold nanoparticles (AuNPs) as an indicator without any modification and labeling step. Two colorimetric logic gates were constructed based on the aggregation of AuNPs mediated by molecular recognition between DNA and protoberberines (palmatine/berberine), including a NIMPLY logic gate and a multi-input NIMPLY + OR logic gate. The logic gate allows the detection of DNA concentrations as low as 0.25 mu g/mL by naked eyes within 10 min. More importantly, a microfluidic device was successfully designed to perform the DNA molecular logic gates for reducing reagent consumption and performing precisely and automatically. The working volume of the logic system has been successfully reduced from 0.5 mL in bulk to 22 mu L in our designed microfluidic chip, and the DNA solution needed was only 1.1 mu L. The method presented here showed great potential in bioanalysis, diagnostics and therapeutics.