Provided in the present invention are a method for constructing a nucleic acid single-stranded cyclic library and reagent kit thereof. The method comprises the steps of using a transposase embedding complex to randomly break nucleic acids and connect a first linker; connecting a second linker at a gap; performing a first PCR reaction, wherein the 5′ end of one of the primers has a first affinity tag, resulting in a product with two ends connected to different linker sequences; binding the product to a solid vector having a second affinity tag; degenerating and separating single strands having no affinity tag; and cyclizing the single strands.