Ligusticum chuanxiong Hort. (L. chuanxiong) is one of the most important herbs in traditional Chinese medicine. It is mainly produced in Sichuan Province, China. Dujiangyan City is considered the authentic production region (APR, superior-quality production region). However, there are differences in the geographical environments and cultivation conditions between the APR and non-APR, which may affect the accumulation of secondary metabolites, cause a difference in bioactivity, and eventually influence the quality of L. chuanxiong. Therefore, antiplatelet aggregation biopotency (AAB) in vitro of L. chuanxiong from different production regions was measured using a parallel-line assay based on the quantitative response method of the bioassay. The result of AAB indicated that the biopotency of L. chuanxiong from Dujiangyan city was stronger than that from other production areas, which scientifically validated Dujiangyan City as the APR of L. chuanxiong. The biopotency of L. chuanxiong from Shifang City was similar to that from Dujiangyan City, indicating that Shifang City could be considered a new APR. The quality markers of L. chuanxiong were analyzed through partial least squares-discrimination analysis based on AAB and 20 active ingredients. Six components were screened as quality markers, where Z-ligustilide, coniferyl ferulate, 3,5-O-dicaffeoylquinic acid were positively correlated with its quality, while cryptochlorogenic acid, butylidenephthalide, and neocnidilide were negatively correlated with it. Among the quality markers, they all exerted antiplatelet aggregation bioactivity, and the high content of Z-ligustilide was the quality feature in the APR. The correlations of the quality markers with the longitude, latitude, altitude were investigated through orthogonal partial least squares. The larger latitude, smaller longitude and higher altitude were beneficial for the accumulation of the quality markers. This may provide a reference for the suitable production regions and quality evaluation of L. chuanxiong.