The invention belongs to the molecular biology technology field, and concretely relates to a method for sequencing and development of Asplenium nidus L. EST-SSR primers based on transcriptome. The method comprises the following steps: firstly, a transcriptome library is constructed; secondly, transcriptome data is obtained, namely, sequencing data is subjected to splicing by utilization of a Trinity software and sequences are spliced into a complete transcriptome; thirdly, SSR site search is carried out, namely, combined with a Perl programming language method, a lot of sequence information is subjected to batch processing, and SSR site search is carried out; fourthly, batch design of EST-SSR primers is carried out. Different fiddlehead materials are selected to verify the designed SSR primers, if strips are detected, successful SSR primers are obtained. Through the method, 4063 pairs of SSR primers are designed, and a new method and thought are provided for development of Asplenium nidus L. EST-SSR primers and further for achieving molecule label auxiliary selection breeding.