Sulfur is one of the inorganic elements used by plants to develop and produce phytoalexin to resist certain diseases. This study reported a method for preparing a material for plant disease resistance. Sulfur nanoparticles (SNPs) stabilized in the chitosan-Cu²⁺ (CS-Cu²⁺) complex were synthesized by hydrolysis of Na₂S₂O₃ in an acidic medium. The obtained SNPs/CS-Cu²⁺ complex consisting of 0.32% S, 4% CS, and 0.7% Cu (w/v), contained SNPs with an average size of ~28 nm as measured by transmission electron microscopy images. The X-ray diffraction pattern of the SNPs/CSCu²⁺ complex showed that SNPs had orthorhombic crystal structures. Interaction between SNPs and the CS-Cu²⁺ complex was also investigated by ultraviolet-visible. Results in vitro nematicidal effect of materials against Meloidogyne incognita showed that SNPs/CS-Cu²⁺ complex was more effective in killing second-stage juveniles (J2) nematodes and inhibiting egg hatching than that of CS and CS-Cu²⁺ complex. The values of LC₅₀ in killing J2 nematodes and EC₅₀ in inhibiting egg hatching of SNPs/CS-Cu²⁺ complex were 75 and 51 mg/l, respectively. These values were lower than those of CS and the CS-Cu²⁺ complex. The test results on the nematicidal effect against M. incognita on coffee pots showed that the SNPs/CS-Cu²⁺ complex was 100% effective at a concentration of 150 mg/l. Therefore, the SNPs/CS-Cu²⁺ complex could be considered as a biochemical material with potential for agricultural applications to control root-knot nematodes.