In this study, a method combining room temperature pulsed laser deposition (PLD) and direct pulsed laser recrystallization (DPLR) is introduced to deposit transparent conductive oxide (TCO) layer on low melting point flexible substrates. Alumina-doped zinc oxide (AZO), as one of the most promising TCO candidates, has now been widely used in solar cells. However, to achieve optimal, electrical, and optical properties of AZO on low melting point, flexible substrate is challenging. DPLR technique is a scalable, economic, and fast process to remove crystal defects and generate recrystallization at room temperature. It features selective processing by only heating up the TCO thin film and preserve the underlying substrate at low temperature. In this study, AZO thin film is pre-deposited by PLD on flexible and rigid substrates. DPLR is then introduced to achieve a uniform TCO layer on these substrates, i.e., commercialized Kapton polyimide film, micron-thick Al-foil, and sold lime glass (SLG). Both finite element analysis simulation and designed experiments are carried out to demonstrate that DPLR is promising in manufacturing high quality AZO layers without any damage to the underlying flexible substrates. The hall mobility of AZO after DPLR on Kapton and SLG reached 198 cm2/v · s and 398 cm2/v · s respectively, while the carrier concentrations are reduced to 2.68 × 1018 and 4.3 × 1019/cm−2, respectively. These characteristics are exactly what an ideal TCO layer should carry: high conductivity and high transmission. The property changes are due to the reduction of defect density after DPLR.