Functionalization of DNA nanostructures is critical to the achievement of the application in biosensors. Herein, we demonstrate a novel DNA three-way junction (TWJ) with three functional moieties, which integrates the electrochemical, fluorescent, and colorimetric properties. Upon addition of external stimuli, including DNA, thrombin, and ATP, the specific interactions between targets and sensing elements could induce strand displacement reaction and conformation transformation, resulting in the integration of G-quadruplex/hemin complex as electrochemical probe, lighting up the fluorescence of DNA/Ag nanoclusters and enhancing the catalytic activity of DNAzyme to catalyze the H2O2-TMB system to generate colorimetric signal. Such a functional DNA nanostructure actually serves as a biosensing platform, showing high sensitivity and selectivity for DNA, thrombin, and ATP detection. Furthermore, We also show that this novel sensing platform can be utilized to detect three different kinds of targets independently and simultaneously. Our integrated concept provides a paradigm for exploring the potential of TWJs in analytical applications.