To clarify the geometric effect of the ultra-fast photocrosslinking reaction of photoreactive oligodeoxyribonucleotide containing 3-cyanovinylcarbazole nucleoside (CNVK) on uridine in complementary RNA strands, pseudouridine (Ψ), which is an isomer of uridine with a C5–C1′ glycosidic bond, was introduced to the photocrosslink site of CNVK in complementary RNA instead of U. The photoreactivity of CNVK toward Ψ was two-fold lower than that of U, suggesting that the geometry between the vinyl moiety on CNVK and the reactive double bond in the pyrimidine base has a large affect on the photoreactivity of CNVK. Contrary to the case of U, the reactivity of the CNVK toward Ψ was decreased by the decrease of reaction temperature below the Tm of heteroduplex, suggesting that the flexible structure of the duplex is advantageous for the photocrosslinking reaction with Ψ, whose reactive double bond possesses unfavorable geometry for the photocrosslinking reaction with CNVK. These basic findings might contribute to the development of a geometry selective photocrosslinking reaction. This is the first example of a sequence specific photocrosslinking reaction toward Ψ, which is the most abundant posttranscriptionally modified nucleoside.