Abstract: Provided is a method for synchronously sequencing a sense strand and an antisense strand of an insert DNA, including: performing two rounds of rolling circle amplification and multiple displacement amplification to obtain a DNA nano ball template including a read1 strand sequencing template and a read2 strand sequencing template; and hybridizing the read1 strand sequencing template and the read2 strand sequencing template with read1 strand sequencing primers and read2 strand sequencing primers, respectively, and simultaneously performing read1 strand sequencing and read2 strand sequencing to obtain sequences of the sense strand and the antisense strand of the insert DNA. The method of the present disclosure can perform the sequencing from both ends of the insert DNA, significantly saving the time and costs for sequencing, and increasing the sequencing throughput.