A recombinant Herpes Simplex Virus type 1 (HSV-1) strain H129-derived anterograde multi-synaptic transneuronal viral tracer for multi-synaptic neural circuit mapping comprises two or more fluorescence expression cassettes being integrated into the H129 genome at different locations； wherein each fluorescence expression cassette contains at least two copies of fluorescent protein-encoding sequence that are arranged in tandem, and at least one linker-encoding sequence, where at least one linker-encoding sequence is disposed between two fluorescent protein-encoding sequences, allowing transcription of fluorescent protein-encoding sequences and linker-encoding sequence as a single transcript； and wherein the linker-encoding sequence encodes a linker peptide containing at least two adjacent amino acids that are highly inefficient in forming a peptide bond between them； thereby, when the single transcript is translated, at least two fluorescent proteins are stoichiometrically generated due to the impedence of peptide bond formation by the linker peptide.
