This paper presents the experimental investigation for the effect of axial heat conduction on the heat transfer analysis in microchannel flow. Molecule-based temperature sensors Rhodamine B/DI water and Ru(bpy)/dope have been respectively applied as temperature sensitive fluid and temperature sensitive paint for fluid and surface temperature measurements in a PDMS microchannel chip with parallel channel design. Under constant heat flux from the floor of microchannel, fluid temperature increases upto 13 degrees C have been observed when Reynolds number varies from 15 to 80. Non-linear fluid and surface temperature variations have been identified by molecule-based temperature sensor measurements. More than half of the temperature increase occurs within 1/8 of channel length from the entrance at a Reynolds number of 15. The acquired temperature data show non-linear variation and deviate from the ideal condition, which is linear, predicated by theoretical analysis. The increase of temperature deviation and the shift of maximum temperature deviation toward the channel entrance have been identified at lower Reynolds numbers. The Nusselt numbers obtained from the experimental data are between 2.55 and 2.58 and these are smaller than those from the theoretical analysis in rectangular channel model (4.84) with constant heat flux from one wall. These deviations are attributed to the axial heat conduction which directs portion of heat toward the entrance and reduces convective heat transfer in the fully developed region. The experimental evidence in this study validates the effects of axial heat conduction in microchannel flow with detailed temperature evolution. (C) 2013 Elsevier Ltd. All rights reserved.