The invention discloses a method for preparing a zero-background cloning vector, and application of the zero-background cloning vector. The method for preparing the zero-background cloning vector comprises the following steps: providing a DNA segment as shown in the sequence SEQ ID NO:6; performing first PCR amplification by taking pUC19 plasmid as a template and utilizing primers as shown in the sequences SEQ ID NO:7-8, thereby obtaining a pUC19 vector segment; performing homologous recombination on the DNA segment and the pUC19 vector segment, thereby obtaining a vector framework; converting the vector framework into competent cells, and extracting the plasmid, thereby obtaining a previous vector of the zero-background cloning vector; and performing enzyme digestion on the previous vector of the zero-background cloning vector by using EcoRV enzyme, thereby obtaining the zero-background cloning vector. The vector obtained by using the method can be directly used for connecting flat tail end products, the vector self-connection background is low, the positive rate of cloning is high, blue and white screening is not needed, rapid and effective cloning can be achieved within a short time, the cloning efficiency is high, and the effect is good.