Examining a greater number of short tandem repeats (STRs) is essential for cases involving mutations, consanguinity, and complex kinship testing. However, previous population studies were mostly limited to fewer than 40 STRs. Therefore, this study investigated the genetic polymorphism, linkage disequilibrium, and mutation rates of 81 autosomal STR loci in the Han Chinese population of northern China. We confirmed the core repeat structure of STR loci, and focused on evaluating the forensic application value of the Category C and 21 additional STR loci not included in the standard “Forensic science-Data structures of selected loci from the DNA database” (GB/T 41009-2021). Blood samples from 400 unrelated healthy Northern Han Chinese individuals and parent-child blood samples from 157 families were detected using GSTAR ^TM25, GSTAR ^TM31FS, and GSTAR ^TM29HS kits. Genetic analysis was performed on genotyping data of 81 autosomal STR loci. The core sequence structures of newly added 15 STR loci were confirmed by sequencing the allelic ladder. After applying Bonferroni correction, all 81 autosomal STR loci conformed to Hardy-Weinberg equilibrium. The observed heterozygosity ranged from 0.4375 to 0.9075, discrimination power from 0.6332 to 0.9845 (lowest at LPL), polymorphism information content from 0.3938 to 0.9092, probability of exclusion for trios from 0.1384 to 0.8108, and for duos from 0.0955 to 0.7069. Significantly, linkage disequilibrium was observed between D5S2500 and D5S2800 loci. When excluding the D5S2800 locus, the cumulative discrimination power (CDP) of remaining 80 autosomal STR loci was 1-2.293×10^-89, cumulative probability of exclusion (CPE) of trios was 1-5.074×10^-31, and cumulative exclusion probability of duos was 1-1.400×10^-19. A total of 13 mutations were observed across 508 meiotic events in 157 families at 10 STR loci. The mutation rates for D5S818, D11S2368 and D18S51 were 0.004, while those of D6S1043, D7S1517, D11S4463, D13S325, D14S1434, D20S482 and D20S85 were 0.002. In conclusion, the 81 genetic loci, including Category C and newly added STRs, exhibit good or moderate polymorphism and are suitable for individual identification, paternity testing, and kinship identification. Given the linkage disequilibrium between the D5S2500 and D5S2800, if simultaneous on the capillary electrophoresis platform, it is recommended to use only the test results from the D5S2500 locus for statistical analysis. This data generated provide allele frequency and mutation rate for a large number of STRs, along with the clarified core sequences, thus offering an important data foundation for DNA evidence evaluation.