This paper reports the shift in thermal stability of DNA duplex and its thermodynamics spectroscopically, caused by stretching and orientation of DNA strands in a microchannel laminar flow. For direct spectroscopic measurement of the microchannel, we prepared an in-house temperature-controllable microchannel-type flow cell. The melting curves of DNA oligomers in a microchannel laminar flow were measured. For DNA oligomers with more than 10 base pairs, the melting curve shifted to the high-temperature side with higher flow speed. However, for 8-base-pair DNA oligomers, a change in the melting profile was not observed in batchwise and microchannel flows. We undertook microfluidic thermodynamic analysis to elucidate details of the shift in thermal stability of the DNA duplex in a microchannel laminar flow. Enthalpy-entropy compensation is applicable to the microfluidic thermal stability shift. We studied the relationships between the enthalpy-entropy compensation and DNA strand length or flow speed. Results showed that the enthalpy-entropy compensation was influenced by both DNA strand length and flow speed, and the penalties of enthalpy were 2-12% greater than the benefits of entropy.