In solid-phase oligonucleotide synthesis, a solid support modified with a universal linker is frequently used to prepare oligonucleotides bearing non-natural- or non-nucleosides at the 3'-end. Generally, harsh basic conditions such as hot aqueous ammonia or methylamine are required to release oligonucleotides by 3'-dephosphorylation via the formation of cyclic phosphate with the universal linker. To achieve 3'-dephosphorylation under milder conditions, we used O-alkyl phosphoramidites instead of the commonly used O-cyanoethyl phosphoramidites at the 3'-end of oligonucleotides. Alkylated phosphotriesters are more alkali-tolerant than their cyanoethyl counterparts because the latter generates phosphodiesters via E2 elimination under basic conditions. Among the designed phosphoramidites, alkyl-extended analogs exhibited rapid and efficient 3'-dephosphorylation compared to conventional cyanoethyl and methyl analogs under mild basic conditions such as aqueous ammonia at room temperature for 2 h. Moreover, nucleoside phosphoramidites bearing 1,2-diols were synthesized and incorporated into oligonucleotides. 1,2,3,4-Tetrahydro-1,4-epoxynaphthalene-2,3-diol-bearing phosphoramidite behaved like a universal linker at the 3'-terminus, allowing dephosphorylation and strand cleavage of the oligonucleotide chain to occur efficiently. Our strategy using this new phosphoramidite chemistry is promising for the tandem solid-phase synthesis of diverse oligonucleotides.