Tio Pakusadewo mulai dikenal oleh publik Indonesia setelah berperan dalam film layar lebar Cinta Dalam Sepotong Roti arahan Garin Nugroho pada tahun 1990, Pada awal tahun 1990-an, tokoh yang masih terhitung kerabat Kadipaten Pakualaman ini dikenal sebagai bintang film muda yang penuh talenta. Berkat perannya sebagai Aria, seorang komponis idealis dalam film Lagu Untuk Seruni (1991), ia meraih Piala Citra di Festival Film Indonesia 1991 untuk kategori Aktor Terbaik. Namun bersamaan dengan tenggelamnya film Indonesia pada pertengahan 1990-an, Tio menghilang. Ia terjebak dalam masalah pribadi dan narkoba. Ia kembali berkarier di dunia perfilman dengan berperan pada film Virgin (2004). Kemudian banyak judul film yang kembali ia bintangi seperti Berbagi Suami (2006), Quickie Express (2007), Lastri (2008), Pintu Terlarang (2009). Pada tahun 2009 ia kembali meraih Piala Citra kategori Aktor Terbaik di Festival Film Indonesia 2009 lewat film Identitas (film) arahan sutradara Aria Kusumadewa.
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