To enhance the efficiency of multi-fragment plasmid construction and overcome the limitations of current techniques, this study introduced a method that utilized the recombination system within Saccharomyces cerevisiae living cell. This approach enabled the simultaneous integration of multiple exogenous DNA fragments with linearized plasmid into a circular plasmid, using the construction of an Agglutinin-like gene knockout vector for Pichia stipitis as an example. The method involved designing primers to introduce 50 bp overlapping sequences between interconnected DNA fragments, followed by PCR amplifying of these fragments. Subsequently, the linearized plasmids were co-transformed with the DNA fragments into Saccharomyces cerevisiae cells. Validation of the plasmids accuracy was then performed in positive yeast colonies through PCR and sequencing. This method offers a high level of precision and considerably reduces the time required for constructing multi-fragments plasmids.