This paper presents a paper-based concentration gradient chip to analyze colorimetric glucose assay. The paper-based concentration gradient chip was fabricated through a wax patterning technique that can design the fluidic channel by selectively printing hydrophobic and hydrophilic areas. Afterwards, glucose and dilution solutions were loaded into the inlet of a concentration gradient chip and each solution was then mixed sequentially at mixing channel. Finally, concentration gradient was formed at each outlet of the chip. To measure the glucose concentration of the solution in outlets, we conducted colorimetric glucose assay with fixed concentration of glucose solution (0, 5, 10, 15 and 20 mM) and obtained normalized intensity. Subsequently, glucose concentrations of the outlets were calculated by substituting the normalized intensity to linear regression function based on the normalized intensity of fixed glucose concentration. Finally, the concentration gradient of glucose was formed on the chip with the result of colorimetric assay. The concentration gradient paper chip has the potential to accurately analyze unknown glucose concentration.