A micro-structured mold-core of a microfluidic chip and its manufacturing method, which includes the steps of: installing a mold-core on a worktable of a five-axis machining center, and installing a micro-milling cutter and a fine milling-grinding tool on a tool holder of the five-axis machining center; rough-milling a surface of the mold-core using the micro-milling cutter according to a preset first machining track, to form a micro-projection array structure with a specific shape; finishing a surface of the micro-projection array structure formed by rough-milling using the fine milling-grinding tool according to a preset second machining track, to form a desired micro-projection array structure; and installing the mold-core on an injection molding machine, and adding particle material of polymer for micro injection molding to form a microfluidic chip, or installing the mold-core on a hot-embossing machine, and adding block material of polymer for hot embossing to form a microfluidic chip.