Against the backdrop of Moore''s Law approaching its limit and the difficulty and surging cost of next-generation integrated circuit technologies, advanced substrate technology is an important carrier to support huge I/O enhancement as well as system integration in the field of advanced packaging, and is one of the core components in the post-Moore era. Currently, semi-additive process based on build-up film (BF) is one of the main ways to realize fine-pitch multilayer packaging substrates. In view of the increasingly prominent problem of signal integrity when electronic equipment operates in high-frequency and high-speed scenes, this paper deeply discusses the influence of physical property of BF materials and structural characteristics on signal transmission loss. Based on typical substrate structures such as microstrip lines and vias, the relationship between BF material parameters and signal transmission performance is studied by electrical simulation analysis system. It is found that in microstrip structure, the signal transmission loss increases with the increase of frequency, and this loss is closely related to the dielectric loss factor of BF material. However, in the via structure, the dielectric constant of BF material has a significant influence on the equivalent capacitance and impedance extreme value, and then affects the impedance mismatch. Although the characteristics of BF material have some influence on impedance mismatch, the design of via structure itself is still the main factor affecting impedance matching. In addition, the conductor loss caused by conductor skin effect increases with the increase of copper foil roughness at high frequency, which provides an important reference for the quality control of copper foil in the manufacturing process of packaging substrate. This study reveals the influence mechanism of BF material and structural characteristics on signal transmission loss, which provides a theoretical basis for the design and optimization of BF material with improved physical properties for packaging substrate.