In this study, the observational arc-length effect on orbit determination (OD) for the Korea Pathfinder Lunar Orbiter (KPLO) in the Earth-Moon Transfer phase was investigated. For the OD, we employed a sequential estimation using the extended Kalman filter and a fixed-point smoother. The mission periods, comprised between the perigee maneuvers (PM) and the lunar orbit insertion (LOI) maneuver in a 3.5 phasing loop of the KPLO, was the primary target. The total period was divided into three phases: launch–PM1, PM1–PM3, and PM3–LOI. The Doppler and range data obtained from three tracking stations [included in the deep space network (DSN) and Korea Deep Space Antenna (KDSA)] were utilized for the OD. Six arc-length cases (24 hrs, 48 hrs, 60 hrs, 3 days, 4 days, and 5 days) were considered for the arc-length effect investigation. In order to evaluate the OD accuracy, we analyzed the position uncertainties, the precision of orbit overlaps, and the position differences between true and estimated trajectories. The maximum performance of 3-day OD approach was observed in the case of stable flight dynamics operations and robust navigation capability. This study provides a guideline for the flight dynamics operations of the KPLO in the trans-lunar phase.