In the past decade, recombinant adeno-associated virus (rAAV) has gained increased attention as a prominent gene therapy technology to treat monogenetic disease. One of the challenges in rAAV production is the enrichment of full-rAAV particles containing the gene of interested (GOI) payload. Herein, we demonstrated that by adjusting the mobile phase properties of anion-exchange chromatography (AEX), Empty and Full separation of rAAV was improved in monolith based preparative AEX chromatography. When compared to the baseline method using NaCl, the presence of tetraethylammonium acetate (TEA-Ac) in the AEX mobile phase resulted in enhanced resolution (from 0.75 to 1.23) between Empty and Full peaks by salt linear gradient elution, as well as increased the percentage of full-rAAV particles from 20% to 36% and GOI genome recovery (from 59% to 62%). Furthermore, a dual wash + step elution AEX method was developed to harness TEA-Ac contribution on Empty and Full separation in the first wash (wash1) step while removing TEA-Ac in the second wash (wash2) step to ensure product safety. The resulting optimized AEX purification method could be easily adapted in scaled-up manufacturing and could also be applied to purification processes involving other AAV serotypes facing similar Empty and Full rAAV separation challenges.