Filament amplification is a key step in artificial seedling cultivation of Scytosiphon lomentaria. In order to optimize the growth conditions of the filament of S. lomentaria, a Box-Behnken design (BBD) was employed for experimental design and analysis of the results to obtain the best possible combination of temperature (X₁: 17–23 °C), light intensity [X₂: 43.2–100 μmol/(m²·s)], and added nitrogen: phosphorus (N∶P) ratio (X₃: 7–17). The results show that the mathematical model has a good fitting degree and good regression property. The model predicted and optimized reaction parameters as follows: temperature 20.45 °C, light intensity 78.13 μmol/(m²·s), added N∶P ratios 16.19. Under the condition of the amplification for 10 d, the increasing ratio of filament biomass was 299.21%±13.58%, which was significantly higher than that measured in the single factor experiment and Box-Behnken design experimental conditions. In order to evaluate the development of filaments of S. lomentaria after optimized culture, the filaments were induced for 20 days. The results showed that the sporangium ratio after optimized culture (26.37%±5.22%) was higher than that of unoptimized culture (18.10%±3.51%). Meanwhile, the average sporangium diameter under optimized conditions was (19.75±0.21) μm, which was also higher than that before optimized culture [(16.91±0.36) μm]. The results showed that the high amplification rate and the development of sporangia of filaments could be achieved by purposeful regulation of environmental conditions and appropriate nutrient concentration.