The Baculovirus Expression Vector System (BEVS) is an insect cell-based heterologous protein expression system that possesses powerful potential in the development of protein drugs and vaccines. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most widely-used vector in BEVS with 151 open reading frames (ORFs) containing essential and nonessential genes. Deletion of nonessential genes has many advantages including increased foreign gene insertion. In this study, the λ red recombination system was used to knock out genes in a modified AcMNPV that carried an enhanced yellow fluorescent protein (eYFP) at the Ac126-Ac127 locus. Eighty genes were almost completely deleted respectively and 69 gene knockout AcMNPVs (KOVs) were obtained to evaluate their infection efficiency. After infecting Spodoptera frugiperda 9 (Sf9) cells, 51 KOVs including 62 genes showed similar infectivity as wide type (WT) and hence were defined as nonessential genes. However, 18 KOVs produced fewer infectious virions, indicating that these genes were influential in the production of progeny viruses. Combining our research with previous studies, a desired minimal AcMNPV genome containing 86 ORFs and all of the homologous regions (hrs) was brought up, facilitating genetic modification of baculovirus vectors and improvement of recombinant protein expression in the future.