To reduce the substrate cost of spermidine synthesis, the feasibility of synthesizing spermidine in vitro from aspartic acid was investigated, and a multi-enzyme cascade catalytic system for in vitro spermidine synthesis was constructed. Through gene editing, aspartate kinase (AK) and aspartic-β-hydroxamate dehydrogenase (ASADH) from Escherichia coli, carboxyspermidine dehydrogenase (CASDH) from Agrobacterium fabrum str. C58, and carboxyspermidine decarboxylase (CASDC) from Campylobacter jejuni 81116 were overexpressed in E. coli to obtain the crude enzyme solution. Spermidine was successfully synthesized in vitro from aspartic acid and putrescine using a multi-enzyme cascade catalytic system. The factors influencing enzyme expression conditions, namely OD600 at induction, final IPTG concentration, induction temperature, and induction duration, were systematically investigated. Concurrently, the enzymatic properties of AK and ASADH, including optimal temperature, thermal stability, optimal pH, and metal-ion dependence, were comprehensively characterized. Using the optimized system, 12.72 mg•L-1 spermidine was ultimately synthesized from 0.44 g•L-1 (5 mmol•L-1) putrescine and 0.665 g•L-1 (5 mmol•L-1) aspartic acid feedstocks. The feasibility of spermidine synthesis from inexpensive aspartic acid via an in vitro multi-enzyme cascade was demonstrated. This study provides valuable insights for further research on the in vitro production of spermidine.