Oligonucleotides libraries have been developed for various applications, but the library size of oligonucleotide increases dramatically with the addition of oligonucleotide length. To assess the possibility of shortening library size by using universal base, deoxyinosine (dITP), the effect of single/multiple dITP substituted in oligonucleotide on ligation was investigated. It was found that different pairs with dITP had different ligation patterns and pairs with dITP at different locations also showed different ligation patterns. With the departure of substitution position from ligation site, the ligation yield increased. Single dITP substitution at ligation site did extremely hurt the ligation efficiency, except for I:C pair. On the other hand, single substitution at two bases or more apart from ligation site, there is no obvious effect on ligation. Multiple dITP substitutions can more or less affect the ligation, besides I:C pair. This research demonstrated that dITP can be applied to reduce oligonucleotide library size after substitution.