Messenger RNA polyadenylation in eukaryotes marks the end of a transcript, and the process is associated with transcription termination. Increasing evidence reveals the potential of gene expression regulation through alternative polyadenylation. The site of poly(A) addition is defined by poly(A) signals reside in the transcribed pre-mRNA. To gain further insight into poly(A) signals and their functions in defining alternative polyadenylation sites that lie within different genomic regions, SignalSleuth2 was developed to extract and analyze cis-elements from a set of data with known poly(A) sites. After obtaining the sequences surrounding the poly(A) sites, exhaustive search of short sequence motifs in specified range of nucleotide sequences are performed, variable motif sizes and rank the detected motifs based on their occurrence frequencies are tallied. It also has new functions including Position-Specific Scoring Matrix (PSSM) scores calculation and multiple scanning modes. This program is powerful in revealing underline sequence motifs surrounding any target regions in a given dataset.