The invention discloses a molecular marker tightly in linkage with main effect QTL of corn line grain number and application thereof. The provided QTL is positioned on a first chromosome 4.09bin, and tightly linked molecular marker consists of primers InDe176 and umc1854. A genome DNA of a triple test-crossed group of corn to be tested is extracted, PCR amplification is carried out by using the primers InDe176 and umc1854, and amplification products having the lengths of 174bp and 142bp are obtained and can be used for predicting the corn line grain number. The molecular marker is used for genotype detection of the corn and its derived varieties or strains to judge the line grain number of the varieties or the strains and improve the selection accuracy and breeding efficiency.