On 25 April 1641, Easter Day, Ambrose and his congregation of around 100 people were surrounded at Morleys Hall, Astley by the Vicar of Leigh and his armed congregation of some 400. Father Ambrose surrendered, and his parishioners were released after their names had been recorded. The priest was restrained, then taken on a horse with a man behind him to prevent his falling, and escorted by a band of sixty people to the Justice of the Peace at Winwick, before being transported to Lancaster Castle.
Father Ambrose appeared before the presiding judge, Sir Robert Heath, on 7 September when he professed his adherence to the Catholic faith and defended his actions. On 8 September, the feast of the Nativity of Mary, Sir Robert Heath found Ambrose guilty and sentenced him to be executed. Two days later, he was taken from Lancaster Castle, drawn on a hurdle to the place of execution, hanged, dismembered, quartered, and boiled in oil. His head was afterwards exposed on a pike.
His cousin, Francis Downes, Lord of Wardley Hall, a devout Catholic rescued his skull and preserved it at Wardley where it remains to this day. It is not the skull of Roger Downes of that same family, the libertine and friend of the Earl of Rochester.
When the news of his death and martyrdom reached his Benedictine brothers at Douai Abbey, a Mass of Thanksgiving and the Te Deum were ordered to be sung.
Canonisation. On 15 December 1929, Pope Pius XI proclaimed Father Ambrose as Blessed at his Beatification ceremony at St. Peter's Basilica, Vatican City. In recognition of the large number of British Roman Catholic martyrs who were executed during the Reformation, most during the reign of Elizabeth I, Pope Paul VI decreed that on 25 October 1970 he was canonising a number of people who were to be known as the Forty Martyrs of England and Wales of whom Ambrose was one.
Hagiography and relics. Challoner compiled Barlow's biography from two manuscripts belonging to St Gregory's Monastery, one of which was written by his brother Dom Rudesind Barlow, President of the English Benedictine Congregation. A third manuscript, titled "The Apostolical Life of Ambrose Barlow", was written by one of his pupils for Dom Rudesind, and is in the John Rylands Library, Manchester; it has been printed by the Chetham Society.
Two portraits of Barlow and one of his father, Sir Alexander, are known to exist.