Four UKIP AMs employ family, as do four Labour and four Tory AMs.
Mr Hamilton said it is the assembly that is responsible for the appointment process, not individual AMs.
"I played no part whatsoever in the appointment of my wife as my PA and diary secretary," Mr Hamilton told BBC Wales on Wednesday.
"She does have 26 years of experience in the House of Commons to fall back on and works 24/7, because it is part of our pillow talk as well."
The former Conservative MP made the comments to The Wales Report programme. It is not against the rules for AMs to employ family members.
"It's totally irrelevant," Mr Hamilton said when asked whether UKIP AMs employing family members sends out a message that they are just like other politicians.
"If you look at the position in the other parties, lots of them do as well.
"In the case of family members it's the assembly that is responsible for the appointments, not the individual AMs.
"They all have to go through a formal selection process carried out by the HR department of the assembly."
According to the current register of members interests, assembly members employing family members are:
The Wales Report, BBC One Wales, 22:40 GMT, Wednesday, 2 November
UKIP assembly group leader Neil Hamilton has said he had no role in the appointment of his wife Christine as his personal assistant.