The invention discloses a sialic acid in bird's nest fragment, and a preparation method and application thereof. The preparation method comprises the following steps: A, soaking and impurity removal: adding bird's nest fragment into water at room temperature for soaking and impurity removal and then carrying out filtering and cleaning; B, drying and crushing: drying the bird's nest fragment and then carrying out crushing and sieving; C, extraction with dilute acid: adding bird's nest fragment powder obtained in the previous step into a dilute acid solution for acid hydrolysis and extraction; D, solid-liquid separation: subjecting an acid solution obtained in the previous step to filtering or centrifugation; E, filtering for impurity removal: carrying out ultrafiltration on supernatant obtained in the previous step and then collecting permeated liquid; F, impurity removal with resin: allowing the permeated liquid to pass through an ion exchange resin column; and G, concentration and drying: subjecting a solution obtained after impurity removal with resin to concentration and drying so as to obtain a sialic acid product. According to the invention, the sialic acid in the bird's nest fragment is converted to a free state, wherein a conversion rate is greater than 99.9%, extraction efficiency is high and the purity of the prepared sialic acid is high; the usage value of cubilose protein is not reduced; and raw materials used in the preparation method are widely available, and the preparation method is suitable for industrial large-scale production.