HAlign is a cross-platform program that performs multiple sequence alignment based on the center star strategy. Here, we present two major updates of HAlign 3, which help improve the time efficiency and the alignment quality. 1) The suffix tree data structure is specifically modified to fit the nucleotide sequence: Left-child right-sibling is replaced by a K-ary tree to build the suffix tree to reach a higher common substring searching efficiency at a small cost in memory usage; 2) a global substring selection algorithm combining directed acyclic graphs with dynamic programming is adopted to screen out the unsatisfactory common substrings. These improvements make HAlign 3 a specialized program to deal with ultra-large numbers of similar DNA/RNA sequences, such as closely related viral or prokaryotic genomes. HAlign 3 can be easily installed via the Anaconda and Java release package on macOS, Linux, Windows subsystem for Linux, and Windows systems, and the source code is available on GitHub (https://github.com/malabz/HAlign-3).