Site-specific integration has emerged as a promising strategy for precise Chinese hamster ovary (CHO) cell line engineering and predictable cell line development. CRISPR/Cas9 with homology-directed repair (HDR) pathway enables precise integration of transgenes into target genomic sites. However, inherent recalcitrance to HDR-mediated targeted integration (TI) of transgenes results in low targeting efficiency, thus requires selection process to acquire targeted integrant in CHO cells. Here we explored several parameters that influence the targeting efficiency using the promoter-trap based single or double knock-in (KI) monitoring system. A simple change in the donor template design by adding sgRNA recognition sequences strongly increased KI efficiency by 2.9–36 fold depending on integration sites and culture mode, compared with conventional circular donor plasmids. Furthermore, sequential and simultaneous KI strategies enabled the generation of double KI populations about 1–4% without the need of additional enrichment processes. This simple optimized strategy not only allowed efficient CRISPR/Cas9-mediated TI in CHO cells but also paved the way for the applicability of multiplexed KIs in one experimental step without the requirement of sequential and independent CHO cell line development procedures.