During the flooding period, agricultural activities in freshwater swamp ecosystems are highly limited. Floating culture is an alternative cultivation system for lettuce production under flooded conditions. This study aimed to determine the effective substrate composition and number of wicks for improving the growth and yield of lettuce (Lactuca sativa L.) in a floating culture system. The experiment was conducted at the Freshwater Swamp Field Laboratory, Sriwijaya University, South Sumatra, Indonesia. The study used a 2.0 m × 1.3 m bamboo raft supported by used mineral water gallons and was arranged in a factorial randomised block design (FRBD) with two factors. The first factor was substrate composition, consisting of M1 = soil, M2 = soil + cow manure + sand (4: 2: 1) and M3 = soil + cow manure + rice husk biochar (4: 2: 1). The second factor was the number of wicks, consisting of S2 = two wicks, S3 = three wicks and S4 = four wicks. Each treatment combination was replicated three times. The results showed that M3 produced the highest fresh leaf weight at 51.60 g and dry leaf weight at 6.13 g, while M1 produced the lowest fresh leaf weight at 10.02 g and dry leaf weight at 1.12 g. The use of four wicks (S4) resulted in the highest fresh leaf weight at 43.51 g and dry leaf weight at 4.68 g. The best treatment combination was M3S4, which produced the highest fresh leaf weight at 61.06 g, dry leaf weight at 7.32 g, fresh stem weight at 1.62 g, dry stem weight at 0.29 g and fresh root weight at 1.69 g. These findings indicate that the combination of soil, cow manure, rice husk biochar and four wicks can improve lettuce yield and may serve as a practical cultivation option for freshwater swamp areas during flooding periods.