The invention discloses a canine parvovirus single-domain antibody, and a preparation method and an application thereof. The canine parvovirus single-domain antibody has a nucleotide sequence as shown in SEQIDNO.1. The preparation method comprises the steps: immunizing an alpaca by a canine parvovirus inactivated vaccine, and taking peripheral blood to obtain alpaca anti-canine parvovirus serum; extracting RNA in the peripheral blood, and carrying out reverse transcription to generate cDNA; utilizing specific primers for amplification, and purifying a VHH fragment; connecting the VHH fragment with a phagemid vector, transforming TG1, and constructing a canine parvovirus single-domain antibody library; and enriching the antibody library by utilizing a canine parvovirus structural protein VP2, introducing into an expression bacterial strain E.coil HB2151, and utilizing IPTG to induce the canine parvovirus single-domain antibody VHH having the biological activity to be expressed. The prepared canine parvovirus single-domain antibody has the detection activity of 15 [mu]g/ml and the detection sensitivity of 250 ng/ml, and can be used as drugs applied for treating dogs with canine parvovirus disease.