The invention discloses a method for yolk antibody-based enzyme-linked immunoadsordent assay (ELISA) detection of turtle systemic sepsis spherical viruses. The method is characterized by comprising the following steps of 1, adding 0.1 nanograms per microliter of turtle systemic sepsis spherical virus antigens into a polystyrene 96-hole reaction plate according to a ratio of 100 microliters per hole, carrying out pre-coating, and carrying out blocking by a bovine serum albumin blocking liquid, 2, adding turtle systemic sepsis spherical virus yolk antibodies diluted according to a certain ratio into each one of holes, and adding horseradish peroxidase-marked goat anti-chicken IgG and an o-phenylenediamine substrate solution into each one of the holes for a color reaction, and 3, reading absorbance values at the wavelength 492 nanometers of all the holes by an ELISA device, and when the absorbance value of the detected hole is more than 2.1 times of an average absorbance value of a negative control hole, determining that the detected result is positive. The method has the advantages of high stability, high specificity, high sensitivity, small immunity interference, and low background signal-noise ratio.