The invention discloses a high-throughput single-cell sequencing library construction method, which comprises the following steps: S1. leading nucleic acid from a single cell to be contacted with a first substance and chain substitute activity-containing polymerase, wherein contact occurs under the condition of the temperature ranging from 4 DEG C to about 25 DEG C; annealing a primer and the nucleic acid, wherein the primer is extended through digestion; and placing the mixture under the condition that the two ends of a total amplicon can be annealed, so that the total amplicon cannot be annealed together with the first substance or a second substance, wherein the second substance contains 30%-40% of G or C; the first substance or the second substance and a semi-amplicon generated by remaining linearity in the mixture can be annealed and extended, wherein the annealing and extension can occur under the condition that nucleic acid cannot be further unwound, and therefore, a total amplicon A generated due to linearity can be generated. The construction method is smart in design and reasonable, and good in effect, high in accuracy for high-throughput sequencing library, and suitablefor population.