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Canine parvovirus (CPV) is a high morbidity and lethality virus which causes severe enteric disease in dogs. In an attempt to gain canine-origin neutralizing antibodies against CPV, single chain variable fragment (scFv) bacteria display libraries against the protective antigen VP2 were constructed and screened. VP2 specific scFvs were selected following three rounds of screening procedures. Selected scFvs were characterized by FCM and ELISA. Seven scFvs showed high affinity and specific binding to CPV. Moreover, the neutralizing activity of the antibody was preliminarily identified by CPV (100 TCID50) in vitro and scFv-2 showed the neutralizing ability with a titer of 32768. This study generated the first canine-origin neutralizing scFv against CPV. The neutralizing scFv would be constructed into full-length antibody in the future. This study laid the foundation for the generation of an effective therapeutic reagent with long half-life and no immunological rejection for the prevention and treatment of CPV infection.