Objective To screen and rescue an influenza A virus strain adapted to suspension MDCK cells in order to provide an experimental basis for the preparation and screening of cell-based influenza vaccine strains. Methods Two influenza virus strains, A/Nebraska/14/2019(H1N1) and A/Guangdong-Maonan/SWL1536/2019＿CNIC-1909(H1N1), were subcultured in suspension MDCK cells for continuous adaptive passaging. One strain stably adapted to suspension MDCK cell culture was screened by detecting hemagglutination titer and cell culture infective dose 50%(CCID_(50)). This strain was used as the parental virus for subsequent reverse genetics manipulation to obtain the influenza backbone strain reH1N1 and reassor-tant strain rcH1N1. The virus particle morphology was observed under electron microscope, and the sequence was verified by plaque purification. The purified recombinant strain rcH1N1 was continuously passaged in suspension MDCK cells,the hemagglutination titer and CCID_(50)were detected, and its genetic stability was verified. Results The hemagglutination titer and CCID_(50)of strain A/Guangdong-Maonan/SWL1536/2019＿CNIC-1909 were more stable and more adapted to suspension MDCK cell culture. The rescued reassortant strain rcH1N1 and the backbone strain reH1N1 had the morphological characteristics of influenza virus particles. The plaque forming units were 8 × 10~7 and 6 × 10~7PFU, respectively, and the PCR product sequences of each gene segment were consistent with those of the recombinant plasmid. The reassortant strain rcH1N1 obtained stable strain with high titer after five passages, the hemagglutination titer reached a maximum of 1∶512, with an lgCCID_(50)of up to 7. 57, and the genetic material of the passage strain remained stable. Conclusion The influenza A virus strain rcH1N1 adapted to suspension MDCK cells was screened and rescued, which lays a foundation for the development of cell-based influenza vaccines.