Abstract: Provided is a method of manufacturing a plastic substrate with lower auto-fluorescence and better specificity. The method includes: (a) preparing a plastic substrate having an Atomic Force Microscopic (AFM) surface roughness of Ra<3 nm or Rq<4 nm under the condition of 50 ?m×50 ?m or less; (b) treating the plastic substrate with plasma; and (c) treating the plastic substrate with a surface-modifying monomer. A plastic substrate manufactured by the method is also provided. The plastic substrate exhibits a remarkably low auto-fluorescence and thus better specificity for detection of target biomolecules, which enables the broad application of a plastic substrate, which can be easily designed to include a microfluidic structure relative to a glass substrate but has been limitedly used due to high auto-fluorescence, to microarrays, biochips, or well plates.