Skipjack tuna samples were collected monthly at Shinkang fishing port, Penghu third fishing market and Penghu Suogang fixed net fishing market from January 2020 to November 2021. The result of the chi-square test showed that there was no significant difference in every months. The result shows that the peak period of spawning in the eastern Taiwan waters is May to July. According to the index of relative importance (IRI), the most important prey species in eastern Taiwan waters were Round scad (Decapterus spp.; IRI = 2409.42), followed by Cephalopods ( Myopsina spp.; IRI = 631.48) and Flyingfish (Exocoetidae spp.; IRI= 502.85). In Penghu samples, the most important prey species were Leiognathus bindus (IRI = 812.49), followed by Spratelloides gracilis (IRI = 388.78) and Benthosema pterotum (IRI = 91.75). The δ15N value was 9.87 ‰, the δ13C value was -17.90 ‰ and the trophic position was 3.78 of skipjack tuna in eastern Taiwan waters. The δ15N value was 10.85 ‰, the δ13C value was -18.02 ‰ and the trophic position was 3.88 of skipjack tuna in Penghu waters. It showed that the δ15N value of Penghu samples was higher than Taitung samples. However, there was no difference between Penghu and Taitung samples in δ13C value. Bayesian mixing model results suggested Cephalopods (Myopsina spp.) as the main prey of skipjack tuna in eastern Taiwan waters (48.4 %), followed by Decapterus macarellus (19.6 %). The most important prey from mixing models for skipjack tuna in Penghu waters were Pterocaesio digramma (26.0 %), followed by Leiognathidae spp. (20.6 %) and Spratelloides gracilis (20.4 %). The complete mitochondrial D-loop sequence was used to analyze the skipjack tuna population along the coast of Taiwan. The results showed that the population of skipjack tuna in Taiwan is quite stable and highly circulated. Haplotype was similar to other studies on NCBI. All samples showed that their genotypes have weak clustering. A total of 110 signs were released in the eastern Taiwan waters this year (38.0 ~ 52.0 cm fork length). Two skipjack tuna recaptured in 14 and 62 days. The other one was recaptured in 440 days.