Biological treatment of food waste (FW) by black soldier fly larvae (BSFL) is considered as an effective management strategy. The composition and concentrations of nutrients in FW change during its storage and transport period, which potentially affect the FW conversion and BSFL growth. The present study systematically investigated the effect of different storage times (i.e., 0–15 d) on FW characteristics and its substantial influence on the BSFL growth. Results showed that the highest larvae weight of 282 mg and the shortest growth time of 14 days were achieved at the group of FW stored for 15 days, but shorter storage time (i.e., 2–7 d) had adverse effect on BSFL growth. Short storage time (i.e., 2–4 d) improved protein content of BSFL biomass and prolonged storage time (i.e., 7–10 d) led to the accumulation of fat content. The changes of substrate characteristics and indigenous microorganisms via FW storage time were the main reasons for BSFL growth difference. Lactic acid (LA) accumulation (i.e., 19.84 g/L) in FW storage for 7 days significantly limited the BSFL growth, leading to lowest larvae weight. Both the substrate and BSFL gut contained same bacterial communities (e.g., Klebsiella and Proteus), which exhibited similar change trend with the prolonged storage time. The transfer of Clostridioides from substrate to BSFL gut promoted nutrients digestion and intestinal flora balance with the FW stored for 15 days. Pathogens (e.g., Acinetobacter) in BSFL gut feeding with FW storage time of 7 days led to the decreased digestive function, consistent with the lowest larvae weight. Overall, shorter storage time (i.e., 2–7 d) inhibited the BSFL digestive function and growth performance, while the balance of the substrate nutrients and intestinal flora promoted the BSFL growth when using the FW stored for 15 days.