We present a novel, dual-color Total Internal Reflection (TIR)-based chip which is capable of generating two overlapping evanescent fields for highly-sensitive and simultaneous detection of two fluorescent materials. We propose a set of governing equations for the overlap condition of two evanescent fields so one can base on to fabricate different chip configurations which still maintain the functionality. The monolithic chip was fabricated using Si bulk micromachining and PDMS casting. Our proposed method integrated all miniaturized components, including two cylindrical microlenses, one prism, two fiber alignment grooves and two fiber stoppers, into one single PDMS chip; thus assembly is unnecessary, and misalignment is avoided. We first demonstrated the capabilities of the chip by detecting simultaneously two fluorescent dyes, namely Tetramethylrhodamine (TMR) and Fluorescein (FI). We then employed the chip to image mixture of Nile-red (NR) and Dragon Green (DG) fluorescent beads. Our miniaturized, integrated device could be an alternative to the conventional dual-color Total Internal Reflection Fluorescent Microscopy (dual-color TIRFM) systems. It could also be a useful component of a micro-Total Analysis System (μ-TAS) for highly-sensitive dual-color fluorescent detection and imaging. © 2008 IEEE.